nÃo hà / A leishmaniose à um grupo de doenÃas com um extenso espectro, cosmopolita, porÃm com ampla e significativa incidÃncia na zona tropical. A leishmaniose apresenta-se sob duas formas, uma visceral envolvendo estruturas do sistema hemolinfopoiÃtico; outra com envolvimento cutÃneo e/ou mucoso, amiÃde, sem visceralizaÃÃo. Esta Ãltima, conhecida como leishmaniose tegumentar (LT), alvo deste estudo, tem como um dos grandes problemas o tratamento, realizado mediante formulaÃÃes injetÃveis com risco de complicaÃÃes no local da aplicaÃÃo e o risco de danos Ãs funÃÃes hepÃtica e renal, alÃm de alteraÃÃes cardÃacas. VÃrios ensaios mostram resultados satisfatÃrios utilizando o FluconazolÂ. Visando a consolidar os resultados descritos na literatura, este estudo procurou demonstrar a eficÃcia terapÃutica do Fluconazol em altas doses no tratamento da LT, em pacientes de uma Ãrea endÃmica no Sul do Estado do CearÃ, Brasil, MunicÃpio de Barbalha. Realizou-se um estudo prospectivo, randomizado, com dois grupos de pacientes. Sessenta do Grupo I fizeram uso de 300mg ou 450mg de Fluconazol durante seis semanas. Sessenta do Grupo II fizeram uso de Glucantime com 20mg/kg/dia, durante 20 dias consecutivos. O diagnÃstico de LT foi realizado com Imprint, cultura, biopsia com histopatolÃgico corado pelo Giemsa e Imuno-histoquÃmica, e a IntradermorreaÃÃo de Montenegro. Todos os pacientes foram avaliados clinicamente e acompanhados durante atà 90 dias. As variÃveis contÃnuas foram avaliadas pelo Test t de Student; para a correlaÃÃo de variÃveis, utilizou-se o Coeficiente de CorrelaÃÃo de Pearson (r). O tempo de cura de cada grupo teve sua avaliaÃÃo realizada pelo mÃtodo de Kaplan-Meier. Em todos os testes de hipÃtese o &#945; foi considerado significante quando menor do que 5% (p < 0,05). A eficÃcia terapÃutica (ET) foi calculada com base na reduÃÃo do risco relativo. A populaÃÃo estudada era predominantemente constituÃda de indivÃduos com faixa etÃria entre 30 e 40 anos de idade, cor de pele parda, rurÃcolas agricultores, com discreta prevalÃncia do sexo feminino. Os resultados mostraram uma eficÃcia terapÃutica de 38,7%. A cura teve relaÃÃo com o tamanho da lesÃo, dados estatatiscamente representativos p< 0,0001. LesÃes com tamanho inferior a 30 mm (isolada ou somatÃrio de vÃrias lesÃes), responderam prontamente ao FluconazolÂ.

During early lactation dairy cows often experience health disorders, which are usually associated with decreased production and reproduction variables. Following parturition, cows use more energy for maintenance and milk production than they consume and enter into a state of negative energy balance. Negative energy balance in early lactation is thought to contribute to decreased milk production, reduced reproductive performance, and increased health disorders. Flunixin meglumine (FM) is an anti-pyretic (fever reducing) and anti-inflammatory drug that is commonly used in the dairy industry. This study evaluated the effect of FM on pyrexia, production and bioenergetic variables in postparturient dairy cows.

The objectives of this study were to evaluate the effects of flunixin meglumine (FM), an inhibitor of PGF2a synthesis, administered at the time of embryo transfer (ET) and insertion of an intravaginal progesterone-releasing device (CIDR) at the time of ET on pregnancy rates (PR) and the resynchronization of estrus. Beef cows (n = 796) and heifers (n = 108) at three locations were assigned randomly within age to one of four groups in a 2 x 2 factorial arrangement of treatments with injection of FM (500 mg i.m.; Phoenix Scientific, St. Joseph, MO) 2 to 12 min prior to ET and insertion of a CIDR (1.38 g progesterone; Pfizer, New York, NY) for 13 d immediately following ET as main effects. Fresh or frozen embryos (Stage = 4 or 5; Grade = 1 or 2) were randomly assigned to be transferred to recipients on d 6 to 9 of the estrous cycle. At Location 2 a subset of fresh embryos were split and transferred as fresh half embryos (n = 192). Recipients at Location 2 only (n = 493) were observed for signs of return to estrus beginning 9 d after ET. Recipients that returned to estrus at Location 2 were either bred by AI 12 h after estrus or received an embryo 7 d after estrus. Pregnancy was diagnosed by ultrasonography. Pregnancy rates were analyzed using the LOGISTIC procedure of SAS. Pregnancy rates of split embryo recipients were analyzed separately using the same statistical procedure. Variation in the timing of the return to estrus was determined by an F-test for heterogeneity of variances. Following the initial ET, pregnancy rates of recipients receiving whole embryos were not affected by CIDR administration (P > 0.05; 65% with CIDR, 70% without CIDR), however, there was a significant FM x location interaction on PR (Location 1, 89 vs. 57%; Location 2, 69 vs. 64%; Location 3, 64 vs. 67% for FM vs. no FM, respectively). There was a significant CIDR x FM interaction on PR of recipients receiving split embryos. Pregnancy rates of split embryo recipients receiving CIDR treatment (54%) or FM treatment alone (41%) were less than controls (64%). However, recipients receiving both CIDR and FM had PR similar to controls (60%). The timing of the return to estrus was more synchronous (P < 0.01) for recipients fitted with a CIDR, but PR of recipients bred following a return to estrus did not differ in cows receiving or not receiving a CIDR (P > 0.13; 68 and 62%, respectively). Effects of FM on PR were location dependent and CIDR insertion at ET improved synchrony of the return to estrus. / Master of Science

Manipulation of the reproductive tract results in increased levels of prostaglandin, which may, in turn, reduce pregnancy rates in embryo recipients. Administration of a prostaglandin inhibitor prior to embryo transfer improves pregnancy rates in cows. Embryo transfer into heifers is more difficult and often requires additional manipulation of the uterus. This study was designed to determine whether administration of the prostaglandin inhibitor, flunixin meglumine, immediately prior to embryo transfer increases pregnancy rates in heifers. Heifers (n=466) were divided into two equal groups based on BCS (range=6-7) and weight (range=256-455). Estrus was synchronized in heifers by giving two injections of prostaglandin F_2" (PGF) eleven days apart with a two day stagger between groups. Heifers in each group were watched for estrus for four days following the second PGF injection. Each heifer detected in estrus (n~389; 83%) was palpated seven days later for the presence and location of an acceptable corpus luteum; development of the reproductive tract (uterine tract score; 1=prepubertal, 5=mature tract) and amount of uterine tone (uterine tone score; l=high tone, 2=medium tone, 3=low tone) were also estimated. The 352 heifers that had an acceptable CL were paired based on day of detected estrus, body condition score, body weight, and uterine tone score. One heifer of each pair was randomly assigned to receive 10ml of flunixin meglumine (IM) just prior to embryo transfer. Time between injection until completion of embryo transfer ranged from 2-25 minutes. All heifers received a single frozen/thawed embryo transferred by one of two experienced technicians. Data collected at the time of transfer included cervix score (1-3; 1=easily penetrated, 3=difficult), ease of transfer score (1-3; l=gun easily manipulated to site of transfer, 3=difficult), embryo placement in the uterine horn (U=upper 1/3, M=middle 1/3, L=lower 1/3), and technician. Pregnancy results were obtained 90 days after transfer via rectal palpation. The logistics procedures and chi-square analysis of SAS were used for data analysis.

Master of Science / Department of Animal Sciences and Industry / John Jaeger / Cattle grazing dormant native range (< 7% crude protein; CP) require supplementation of additional protein to sustain body weight (BW) and body condition score (BCS). Daily delivery of these supplements is an economic burden to cattle producers faced with challenging economic circumstances. Supplementing cows infrequently (as little as once/week) has produced equivalent BW and BCS changes compared to daily delivery. Dried distiller’s grains with solubles (DDGS) provides more ruminally-undegradable protein (RUP; 50-60%) compared to traditional oilseed-meal supplements (i.e. soybean meal) that are >50% ruminally-degradable protein (RDP). Therefore, our objective was to evaluate the effects of supplementation frequency on performance, reproductive success, eating behavior, and subsequent calf performance of spring-calving cows supplemented with DDGS. No differences in ending BW (P = 0.69) and BCS (P = 0.49), or changes in BW and BCS over the supplementation period (P = 0.82 and 0.70, respectively) were observed among cows supplemented every d, every 3 d, or every 6 d. Calf BW at birth, weaning weight (WW), and average daily gain (ADG) were similar among treatments (P = 0.19, 0.12, and 0.10, respectively). First-service conception rate (FSCR) and final pregnancy rate (PR) were also not affected by supplementation frequency (P = 0.62 and 0.76, respectively). The development of replacement heifers is a large expense for cow-calf producers. Improved breeding and heifer development strategies aimed at ensuring the success of replacement females have been developed but reproductive failure still remains a problem. The stress associated with breeding and handling procedures may decrease reproductive success. Therefore, the objective was to determine if intramuscular administration of flunixin meglumine (1.1 mg/kg BW) 14 days post-breeding would improve FSCR and PR in non-transported replacement heifers. Under the conditions of our study, flunixin meglumine did not improve (P = 0.87) first service conception rate above that of control heifers (41.2% and 42.3%, respectively). Final pregnancy rate also was not different between treatments and averaged 81.8% (P = 0.40).

Licentiatavhandling (sammanfattning) Linköping : Linköpings universitet, 2009. / Härtill 2 uppsatser.

[pt] A administração de antimoniais no tratamento da leishmaniose é uma rara oportunidade para estudar o metabolismo de antimônio e das suas espécies químicas no corpo humano. No presente trabalho, a técnica de espectrometria de massa (ICPMS), acoplada ou não à cromatografia iônica (IC) e à geração de hidretos (HG), foi utilizada para determinação das concentrações de antimônio total e de suas espécies químicas, Sb(III), Sb(V) e trimetilantimônio (TMSbV) em amostras clínicas de pacientes com leishmaniose e/ou de macacos Rhesus (Mucaca mulatta) tratados com antimoniato de meglumina (AM). Diferentes regimes terapêuticos foram avaliados, incluindo a administração de doses baixa e alta (5 mg ou 20 mg de Sb(V) por kg de massa corpórea). A concentração total de Sb foi determinada após a decomposição de amostras por HNO3/H2O2. Análises de especiação foram realizadas em amostra de plasma e urina, através do acoplamento em linha da CI ao instrumento de ICPMS. Duas colunas de troca aniônica PRP-X100 foram utilizadas para a separação de espécies empregando o EDTA (eluição isocrática: 4,7 mmol L-1, 2,5% v/v metanol, pH 4,7) ou tampão de EDTA/fosfato (eluição com variação abrupta de eluente: 1o. eluente - 20 mmol L-1 EDTA + 2 mmol L-1, pH 4,5; 2o. eluente - 50 mmol L-1 (NH4)2PO4, pH 8,3) como fase móvel. As características de desempenho de todos os métodos foram avaliadas e serão apresentadas. Testes de estabilidades mostraram que as espécies de antimônio estudadas são suficientemente estáveis permitindo análises de especiação dentro de 24 horas após a coleta. Em humanos, assim como em macacos Rhesus, as concentrações de antimônio em amostras de urina e/ou plasma coletadas após o período de administração de AM, mostraram uma rápida cinética inicial de excreção (t1/2 ~ 3 dias) da droga, seguida de duas fases mais lentas. Determinou-se concentrações de Sb significativamente maiores em frações de hemácias do que nas amostras de plasma correspondentes, com exceção da fase inicial de administração da droga (primeiras 12 horas). A bioredução de Sb(V) a Sb(III) foi observada e confirmada como um importante processo metabólico durante a fase de eliminação. Nenhuma evidência de formação de TMSb (V) foi obtida em nossos estudos. As concentrações de antimônio em amostras de tecidos de macacos Rhesus coletadas aproximadamente 60 dias após a última administração de AM foram maiores na tireóide, seguida por fígado e baço. As concentrações no fígado foram pelo menos 1000 vezes maiores que a concentração basal. Amostras de cabelos/pelos e unhas de pacientes e símios tratados com AM também apresentaram altas concentrações, as quais corresponderam ao histórico de administração da droga e também mostraram que a incorporação de Sb nestes tecidos é acumulativa, mesmo após o fim da administração de AM. Em cabelos, mesmo após mais de 300 dias do fim do tratamento as concentrações de Sb não retornaram ao nível basal. / [en] Clinical applications of antimonials in the treatment of leishmaniasis are a unique opportunity to investigate the metabolism of antimony and its species in the human body. In this work, inductively coupled plasma mass spectrometry (ICPMS), without or in combination with ion chromatography (IC) and flow injection hydride generation (FI-HG), has been used for the determination of total antimony concentrations [Sb] and three of its expected species, Sb (III), Sb (V) and trimethyl antimony (TMSbV), in clinical samples of leishmaniasis patients and/or of rhesus monkeys (Mucaca mulatta) treated with meglumine antimoniate (AM). Different drug administration schemes were evaluated, including low and high dose administration (5 mg or 20 mg of Sb5+ per kg of body mass). Total [Sb] was assayed after wet decomposition of the samples by HNO3/H2O2. Speciation analysis was performed on plasma and urine samples using IC on-line coupled to the ICPMS instrument. Two PRP-X100 anion exchange columns were used for species separation employing EDTA (isocratic elution: 4.7 mmol L-1, 2.5% v/v methanol, pH 4.7) or EDTA/phosphate buffer (step elution: 1st eluent - 20 mmol L-1 EDTA + 2 mmol L-1 KHP, at pH 4.5; 2nd eluent - 50 mmol L-1 (NH4)2HPO4 at pH 8.3) as the mobile phases. Performance characteristics of all methods were evaluated and are presented. Stability tests showed that all studied antimony species are sufficiently stable to allow speciation analysis within 24 hours after collection. In humans, as well as in rhesus monkeys, the concentrations of antimony in urine and/or plasma samples measured after the administration period of MA, showed rapid excretion kinetics (t1/2 ~ 3 days) of the drug followed by two slower ones. Significantly higher concentrations of [Sb] were measured in erythrocyte fractions than in corresponding plasma samples, excepting the very initial phase of drug administration (first 12 hours). Bio-reduction of Sb5+ to Sb3+ was observed and confirmed as an important metabolic process during the slow elimination phase. No evidence for the formation of TMSbV was so far obtained in our studies. Antimony concentrations in tissue samples of rhesus monkeys 60 d after the last AM administration were highest in thyroid, followed by liver and spleen. Liver concentrations were at least 1000 times the basal concentrations. Hair and nail samples from treated patients and monkeys had also very high Sb concentrations, which matched drug administration history and showed also that incorporation of Sb into these tissues is continuing, even after ceasing AM application. In human hair, more than 300 days are required for the return of antimony concentrations to basal levels.

INTRODUÇÃO: O Gadolínio (Gd) é um raro metal da classe dos lantanídios usado como meio de contraste devido as suas propriedades paramagnéticas. Após sua descoberta, foi considerado um contraste pouco nefrotóxico em pacientes com doença renal crônica (DRC). Atualmente, existem evidências de que o Gd pode apresentar nefrotoxicidade semelhante aos contrastes iodados. A administração de Gadolínio em ratos com DRC pode levar a piora da função renal aferida por clearance de inulina e mobilização do ferro corporal gerando stress oxidativo. OBJETIVOS: O objetivo do estudo foi avaliar o efeito do Gd na função renal, nos parâmetros de cinética do ferro em ratos com DRC e o possível efeito protetor do anti-oxidante N-Acetilcisteína (NAC). MATERIAIS E MÉTODOS: Ratos Wistar machos foram submetidos à nefrectomia 5/6 (Nx) para induzir DRC. Gadoterate Meglumine, um contraste à base de Gadolínio iônico e macrocíclico foi administrado via intravenosa na dose de 1,5mmol/kg de peso de rato 21 dias após a nefrectomia. Para avaliar o efeito do Gd sobre a função renal, estudos de clearance de inulina foram realizados em 4 grupos de ratos 48 hs após a aplicação do Gd: grupo controle 1- Nx (n=7); 2- Nx+NAC (n=6); 3- Nx+Gd (n=8); 4- Nx+Gd+NAC (n=5). O NAC foi administrado no grupo 4 diluído em água 48 hs antes e 48 hs depois da administração do Gd na dose de 4800mg/l. O grupo 2 recebeu NAC durante o mesmo período de tempo do grupo 4. O Gd também foi administrado em ratos com função renal normal, grupo Normal (n=8) na mesma dose dos grupos nefrectomizados com avaliação da função renal e proteinúria. Além da taxa de filtração glomerular (TFG), foram avaliados: proteinúria de 24hs (ptn), parâmetros de gaiola metabólica, pressão arterial (PA), paramêtros de cinética do ferro representados pelo ferro sérico (Fe), capacidade total de ligação do ferro (CTLF), saturação de transferrina, ferritina sérica e stress oxidativo por meio da dosagem de espécies reativas ao ácido tiobarbitúrico (TBARS). Os dados foram submetidos à análise de variância ANOVA utilizando o programa Graph Prism com nível de significância p<0,05 e erro padrão. RESULTADOS: A aplicação de Gd em ratos nefrectomizados resultou em queda na TFG no grupo 3 em relação ao grupo controle 1 (p<0,01). Houve uma tendência de aumento da ptn no grupo 3 em relação aos demais grupos. O grupo 4 que recebeu tratamento com NAC apresentou TFG e ptn semelhante ao grupo 1 e TFG estatisticamente maior que o grupo 3 (p<0,05). Com relação ao grupo de ratos normais não houve alteração da TFG nem aumento de ptn após a aplicação do Gd em relação à ratos não nefrectomizados que não receberam contraste. Com relação aos parâmetros da cinética do ferro, o grupo 3 apresentou elevação da ferritina e da saturação da transferrina comparados ao grupo 1 (p<0,05) e (p<0,01) respectivamente. Houve diminuição da capacidade total de ligação do ferro (CTLF) no grupo 3 comparado ao grupo 1(p<0,01). O uso profilático do NAC no grupo 4 reverteu todas as alterações descritas anteriormente no grupo 3 com significância estatística (p<0,05), (p<0,01) e (p<0,01) respectivamente. Com relação ao stress oxidativo, o grupo 3 apresentou níveis de TBARS significativamente maiores que o grupo 1 (p<0,05). O grupo 4 apresentou níveis de TBARS semelhantes ao grupo 1 e menores que o grupo 3 (p<0,05). O grupo 2 que recebeu apenas NAC por curto período de tempo apresentou TFG, Ptn e parâmetros de cinética de ferro semelhantes aos grupos 1 e 4. Com relação aos dados de gaiola metabólica e pressão arterial não houve diferença estatística entre os grupos 1,2,3 e 4. CONCLUSÕES: Os resultados mostram que a administração de Gd em ratos nefrectomizados resulta em dimuição da TFG, aumento da proteinúria associado a aumento da ferritina sérica, saturação de transferrina e diminuição da CTLF. Em ratos normais o Gd não mostrou ser nefrotóxico e o uso do NAC isoladamente no grupo 2 por curto período de tempo não demonstrou nenhum efeito, pois todos os parâmetros avaliados foram semelhantes ao grupo controle. Indução de stress oxidativo foi representado pelo aumento do TBARS nos ratos que receberam o Gd. O uso de NAC reverteu todas as alterações provocadas pelo Gd. Concluímos que o NAC pode ser usado como profilaxia da toxicidade associada ao Gd. / INTRODUCTION: Gadolinium (Gd) is a rare class of lanthanide metal used as a contrast agent due to its paramagnetic properties. After its discovery, was considered a bit of contrast nephrotoxicity in patients with chronic kidney disease (CKD). Currently, there is evidence that Gd may present similar to iodine contrast media nephrotoxicity. The administration of Gadolinium in rats with CKD can lead to worsening renal function measured by inulin clearance and mobilization of iron body causing oxidative stress. OBJECTIVES: The aim of this study was to evaluate the effect of Gd on renal function, iron parameters and oxidative stress in rats with CKD and a possible effect of antioxidant N-Acetylcisteine (NAC). MATERIALS AND METHODS: Male Wistar rats underwent nephrectomy 5/6 (Nx) to induce CKD. Gadoterate meglumine, a Gadolinium based contrast ionic and macrocyclic was administrated intravenously at a dose of 1.5 mmol / kg BW of rats 21 days after nephrectomy. To evaluate the effect of Gd on renal function, inulin clearance studies were performed in 3 groups of animals 48 hours (hs) after application of Gd: a control group 1 - Nx (n =7), 2- Nx+NAC (n = 6); 3- Nx+Gd (n=8) e 4- Nx+Gd+NAC (n=5). The NAC was administrated in group 4, diluted with water 48 hs before and 48 hs after administration of gadolinium at a dose of 4800mg / l. Group 2 received NAC four days before clearance study. Gd was also administrated in rats with normal renal function, group normal (n = 8) at the same dose of nephrectomized rats with assessment of renal function and proteinuria. In addition to the glomerular filtration rate (GFR) were evaluated: 24 hours proteinuria (ptn), cage metabolic parameters, blood pressure (BP), serum iron (Fe), total capacity of iron binding (TIBC), transferrin saturation, serum ferritin and oxidative stress through measurement of thiobarbituric acid reactive species (TBARS). Data were submitted to ANOVA using the program Prism Graph with a significance level p <0.05 and standard error. RESULTS: Gd administration to group 3 results in a decrease of GFR compared with group 1 (p<0,01). There was a trend of increase ptn in group 3 compared to other groups. Normal rats treated with the same dose of Gd presented similar GFR and proteinuria when compared with normal controls. In group 3, there was a decrease in TIBC, elevation of ferritin serum levels, transferrin oversaturation and plasmatic TBARS elevation compared with group 1 (p<0,01), (p<0,05), (p<0,01) and (p<0,05) repectively. The treatment with NAC in group 4 reversed the decreased in GFR and proteinuria compared with group 3 (p<0,05) for both variables. Treatment with NAC in group 4 also reversed all alterations in iron parameters and oxidative stress described earlier with statistical significance (p<0,01), (p<0,05), (p<0,01) and (p<0,05) respectively. Group 2 received NAC for a short period of time had GFR, Ptn and kinetic parameters of iron similar to groups 1 and 4. With respect to metabolic cage data and blood pressure showed no statistical difference between groups 1,2,3 and 4.CONCLUSIONS: These results show that Gd administration to nephrectomized rats results in a decrease of GFR and increased proteinuria associated with increased serum ferritin and transferrin saturation with decreased TIBC. In normal rats, Gd was not nephrotoxic. These effects were not due to a possible effect of NAC on Nx, since all parameters measured in group 2 were not different from the group 1. There was induction of oxidative stress represented by the increase in TBARS in rats receiving gadolinium. The use of NAC reversed all these changes caused by Gd. We conclude that the NAC can be used as prophylaxis of toxicity associated with Gd.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-21T18:53:49Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) GABRIELA TAGLIANI LOPEZ.pdf: 1929634 bytes, checksum: 65b59bcc7ee2dd96fa3d6a96b37b9882 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2016-09-21T18:54:04Z (GMT) No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) GABRIELA TAGLIANI LOPEZ.pdf: 1929634 bytes, checksum: 65b59bcc7ee2dd96fa3d6a96b37b9882 (MD5) / Made available in DSpace on 2016-09-21T18:54:04Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) GABRIELA TAGLIANI LOPEZ.pdf: 1929634 bytes, checksum: 65b59bcc7ee2dd96fa3d6a96b37b9882 (MD5) Previous issue date: 2015-08-14 / A leishmaniose é uma doença infecciosa que pode acometer a pele, as mucosas e os órgãos internos. Apesar desta doença atingir, anualmente, cerca de 1,5 milhão de pessoas em todo o mundo, ainda possui poucas alternativas terapêuticas. O fármaco recomendado pela Organização Mundial de Saúde (OMS) para o tratamento das leishmanioses é o antimoniato de N-metilglucamina (Sb-AMG). O tratamento com esse fármaco pode induzir o indivíduo a um quadro de toxicidade, já que é derivado do antimônio (Sb), um metal encontrado livremente na crosta terrestre, principalmente sob a forma trivalente e pentavalente de oxidação. A forma trivalente mostrou-se clastogênica e com potencial cancerígeno tanto in vitro quanto in vivo. Dentro dessa problemática, estudos envolvendo o Sb-AMG são escassos, levando-se em consideração parâmetros genotóxicos. Este trabalho, portanto, teve como objetivo, avaliar comparativamente os efeitos de cinco diferentes concentrações (5-50 μg/mL) de Sb-AMG e de SbV sobre parâmetros citotóxicos, genotóxicos e mutagênicos em leucócitos humanos. Para os testes de citotoxicidade e genotoxicidade aplicamos a análise estatística de variância (ANOVA) de uma via e para os testes de mutagenicidade a ANOVA de duas vias. Entre as múltiplas comparações utilizamos o teste de Tukey e consideramos um resultado estatisticamente significativo quando p<0,05. Testes de proliferação celular, instabilidade cromossômica, teste do cometa alcalino e ensaio de quebra de fita dupla de DNA, demonstraram que ambas as formas de Sb testadas foram capazes de induzir alterações genotóxicas significativas, quando comparado ao controle negativo em, pelo menos, uma das concentrações ensaiadas. Os testes de viabilidade celular e micronúcleo foram utilizados para avaliar, respectivamente, a citotoxicidade e a mutagenicidade. Adicionalmente, foi avaliado o número de células apoptóticas e necróticas, bem como o índice de divisão nuclear citotóxico. Todos esses parâmetros demonstraram significantes alterações. De uma forma sem precedentes, o nível de Sb intracelular foi quantificado e relacionado com as diferentes concentrações de exposição e, a partir disso, nós sugerimos que o influxo de Sb ocorre de forma concentração dependente, mas com cinética de ordem zero. Tomando conjuntamente os dados obtidos, os resultados evidenciam claramente que o Sb-AMG possui efeitos citotóxicos, genotóxicos e mutagênicos em leucócitos humanos nas concentrações testadas. / Leishmaniasis is an infectious disease that can affect the skin, mucosa and internal organs. Although affecting about 1.5 million people each year, leishmaniasis has few therapeutic alternatives. N-methylglucamine antimoniate (Sb-GAM) is the drug of choice to treat leishmaniasis according to the World Health Organization. The treatment with this drug can induce the individual to a frame of toxicity, since it is derived from the antimony (Sb), a metal free found in the earth's crust, principally in the form trivalent and pentavalent oxidation. The trivalent form has clastogenic and carcinogenic potential both in vitro and in vivo. However, there are few genotoxic studies involving Sb-GAM. The main contribution of this study was to evaluate in vitro the cytotoxic, genotoxic and mutagenic parameters of five different concentrations (5, 10, 20, 40 and 50 μg/mL) of Sb-GAM and SbV in human leukocyte. For the cytotoxicity and genotoxicity tests we apply statistical one-way analysis of variance (ANOVA) and for mutagenicity tests the two-way ANOVA. Among the multiple comparisons we used the Tukey test and consider a statistically significant result when p <0.05. Cell proliferation, chromosomal instability, the alkaline comet assay and double- stranded DNA breaks assays, showed that both forms of Sb were genotoxic when compared to the negative control, at least, in one of the concentrations tested. Cell viability and micronucleus tests were used to evaluate, respectively, cytotoxicity and the mutagenicity. Additionally, there was assessed the number of apoptotic and necrotic cells, as well as nuclear division cytotoxicity index. All these parameters showed significant changes. In an unprecedented strategy, the intracellular Sb level was quantified and correlated with the different exposure concentrations and, from this, we could induce that the Sb inflow occurs in a concentration-dependent, but with a zero-order kinetics. Altogether, our results show clearly that Sb-GAM has cytotoxic, genotoxic and mutagenic effects on human leukocytes at the concentrations tested.

According to WHO, leishmaniasis is endemic in 98 countries, and has been placed ninth in a global analysis of infectious diseases. Treatment of leishmaniasis is based on pentavalent antimonials but toxicity and developing resistance have been reported. Traditional medicine and scientific studies have shown that the extract of Galipea longiflora Krause (Evanta) exhibits antileishmanial activity. We hypothesized that the healing observed when using this plant might not only be due to the direct action on the parasite, but possibly to a parallel effect on the host immune response. We found that an alkaloid extract of Evanta (AEE) inhibited the growth of Leishmania braziliensis promastigotes while viability of eukaryotic cells was practically not affected. We also found that AEE interfered with polyclonal activation or Leishmania-specific re-stimulation of lymphocytes, as revealed by a reduction of in vitro cellular proliferation and IFN-g production. More important, AEE treatment of mice hosting L. braziliensis showed that AEE is able to control both inflammation and parasite load. Additionally, the healing process was improved when AEE and meglumine antimoniate were administered simultaneously. Dendritic cells (DCs) play a pivotal role in T-cell stimulation and polarization of naïve T cells. Therefore, we investigated if AEE could alter the activation of DCs and if allostimulatory DCs properties were altered if activated in the presence of AEE. DCs activated in the presence of AEE reduced the production of IL-12p40 and IL-23. When we analyzed the allostimulatory capacity of AEE-treated DCs, we found that allogeneic CD4+ T-cells secreted lower levels of IFN-γ. In conclusion, this thesis provides valuable insight into the effects of Evanta derived extract. The dual effect found for AEE, on Leishmania parasite and on the immune response, suggests that AEE may be useful in controlling the parasite burden and preventing over-production of inflammatory mediators and subsequently avoiding tissue damage. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Accepted. Paper 3: Submitted.</p>