Introdução: A premissa do programa nacional de controle da leishmaniose visceral (LV) é que a doença humana esteja relacionada com a canina, sendo o controle do reservatório canino um dos focos do programa. Objetivos: Mensurar a taxa de incidência da LV em humanos, relacionando-a com as coberturas do controle químico e do reservatório canino. Relacionar a soroprevalência canina com características dos cães e de seus tutores. E avaliar a eficácia da eutanásia de cães soropositivos no controle da infecção canina. Métodos: Os casos humanos e os domicílios com cães soropositivos registrados no período de 2007 a 2015 no município de Araçatuba, estado de São Paulo, foram geocodificados e calculadas a soroprevalência canina, a taxa de incidência humana e as coberturas das atividades de inquérito sorológico, eutanásia e controle químico. A associação entre as variáveis foi avaliada por comparação de mapas, por regressão linear e pela função K de Ripley. Um estudo transversal foi conduzido entre 2015 e 2016, nos municípios de Araçatuba e Birigui, tendo como base uma amostra da população canina. A soroprevalência foi modelada por regressão logística em uma abordagem geoestatística usando a aproximação de Laplace integrada aninhada para inferência bayesiana. Dados secundários e dos inquéritos realizados foram utilizados para elaborar e calibrar modelos dinâmicos. Resultados: Observou-se no município de Araçatuba a diminuição da taxa de incidência LV e da soroprevalência canina, apesar das coberturas de controle terem sido baixas no mesmo período estudado. O inquérito sorológico revelou uma soroprevalência canina de 8% em Araçatuba e 4% em Birigui. Em Araçatuba a ocorrência de cães soropositivos foi associada à domicílios que tiveram mais de 10 cães ao longo do tempo, domicílios com histórico de cães com a infecção ou que morreram por outras causas não naturais, e à permanência dos cães no peridomicílio ao longo do dia. Foi observada dependência espacial (46 m) entre as observações. Considerando controle contínuo e um esforço das atividades de inquérito sorológico três vezes maior que a média do observado em Araçatuba e duas vezes maior em Birigui, as atividades relacionadas à eutanásia de cães com diagnóstico positivos possibilitariam, em teoria, o controle da infecção canina. Conclusões: A diminuição da LV ao longo do tempo está relacionada com as ações de controle, uma vez que pequenas alterações na dinâmica da infecção canina têm importância epidemiológica. O encontro de dependência espacial entre os domicílios com casos caninos em pequenas distâncias reforça a existência de um padrão local da transmissão da infecção entorno dos domicílios, relacionado com as características do vetor. A eutanásia de cães soropositivos, em teoria, é capaz de controlar a infecção canina, porém, este resultado desse ser entendido com cautela, dada a complexidade operacional desta medida e as questões éticas relacionadas. Novos estudos precisam ser desenvolvidos para uma melhor compreensão se fatores além das atividades de controle estariam envolvidos na diminuição da incidência da LV. Faz-se necessário planejamento a longo prazo das ações de controle e investimento em pesquisas sobre o custo-efetividade de outras medidas que auxiliem no controle da LV. / Background: The assumption of visceral leishmaniasis (VL) control program is that human disease (HVL) is related to canine infection (CanL), and that supports the culling of infected dogs. Objectives: Estimate the coverage of visceral leishmaniasis (VL) control measures and to relate them to the occurrence of HVL in an endemic urban area (Araçatuba, SP). Determine the CanL seroprevalence and to evaluate its relationship with the characteristics of dogs and their owners. Evaluated the culling dogs efficacy in controlling the canine infection. in the municipalities of Araçatuba and Birigui, state of São Paulo, Brazil. Methods: The cases of HVL and households with seropositive dogs registered in the period from 2007 to 2015 were geocoded the coverage of the serological inquiry, culling dogs and chemical spaying, canine seroprevalence and HVL incidence rates were calculated. The relationship between CanL, HVL and control measures was evaluated by map comparison, by linear regression and was also assessed using the Ripley K function. A cross-sectional study was conducted between 2015 and 2016, based on a sample of the canine population. Seroprevalence was modelled by logistic regression in a geostatistical approach using the integrated nested Laplace approximation for Bayesian inference. The spatial component was modelled by a Gaussian field, using a stochastic partial differential equation approach. Secondary data and the surveys were used to elaborate and calibrate dynamic models of canine disease transmission. Results: Verified the decrease of HVL and the CanL in Araçatuba over time, even in low coverage of control measures. The CanL seroprevalence was 8% in Araçatuba and 4% in Birigui. The occurrence of a seropositive dog in Araçatuba was associated with the presence of more than 10 dogs living in the same house, house with dogs that previously died of VL or died of other unnatural causes, and the place of dogs stayed during the day. Spatial dependence among observations occurred within about 46 m. Considering a continuous control and a serological survey effort three times higher than the average of the observed in Araçatuba and twice as high in Birigui, the activities related to culling dogs is effective in controlling canine infection. Conclusions: The observation of VL decrease over time may be related to control measures. The short-distance spatial dependence could be related to the vector characteristics, producing a local neighbourhood VL transmission pattern. The culling dogs were effective in controlling the disease in the dogs\' population. However, this result should be understood with caution given the operational complexity of this measure and related ethical issues. New studies need to be developed for a better understanding if factors other than control activities would be involved in reducing the incidence of VL. It is necessary in addition to long-term planning of the control actions carried out by the program, investment in researches that evaluate the cost-effectiveness of other measures that may help in the control of VL.

The vector-borne protozoan Leishmania spp. cause the spectrum of disease known as leishmaniasis in human and animal hosts. The most common manifestations of leishmaniasis are the chronic, ulcerative skin disease cutaneous leishmaniasis (CL), and the more serious visceral leishmaniasis (VL) in which parasites take up residence in internal organs, causing death if not treated. The role of neutrophils (PMNs) in the immune response to CL and VL is unclear. It is s generally thought that PMNs are only a short-lived effector cell, and have been disregarded as playing a role in chronic Leishmania spp. infection. As both CL and VL are diseases characterized by increased inflammatory immune mediators, we hypothesized that PMNs from human or animal models of chronic leishmaniasis would display different properties from PMNs from healthy controls. We found in a subset of CL and VL patients circulating PMNs expressing HLA-DR, the human form of MHC class II, a molecule thought to be restricted primarily to professional antigen cells. When we examined PMNs recruited to CL skin lesions in human patients, or similar lesions in experimental murine model of CL, we found significantly increased MHC class II+ PMNs. Circulating HLA-DR+ PMNs also expressed the co-stimulatory molecules CD80, CD86 and CD40. While this suggested an antigen-presenting cell-like phenotype by these HLA-DR+ PMNs, compared to conventional HLA-DR- PMNs, HLA-DR+ PMNs showed not only a neutrophil-like appearance and function, but in fact increased activation, degranulation, intracellular MPO and phagocytosis of parasites and zymosan particles. Incubation of healthy control whole blood with inflammatory cytokines resulted in increased HLA-DR+ PMNs and the presence of hladrb1 mRNA, suggesting a connection between neutrophil “priming” and upregulation of HLA-DR. In addition to HLA-DR+ PMNs in CL patients, we also identified the presence of so-called “low-density” neutrophils (LD-PMNs). These neutrophils, which are enriched in low-density fractions following centrifugation of blood over a density gradient, are reported in numerous disease states, including cancer, HIV, and systemic lupus erythematosus. In some disease states, LD-PMN are reported to be immunosuppressive toward T cell activation and proliferation. However, LD-PMNs from leishmaniasis patients showed no evidence of immunosuppression. Additionally, we found that LD-PMNs show significantly increased surface expression of MHC class II, suggesting a heretofore unappreciated connection between these atypical neutrophil phenotypes. We also investigated the in vitro interactions with different Leishmania infantum life-stages, both those that cause acute infection (promastigotes) and amastigotes, which are found during chronic stages of the disease. We found that PMNs are readily infected by all L. infantum life-stages, but that amastigotes may have different methods of interacting with PMN surface receptors and are better equipped to avoid PMN anti-microbial responses. These data suggest that circulating PMNs in chronic leishmaniasis may have unique phenotypes and interact differently with the Leishmania spp. life-cycle present during chronic infection. Further investigation of the role of PMNs and atypical PMN phenotypes in chronic disease may help identify new immunomodulatory roles for this cell type.

Leishmaniasis is a parasitic disease that affects humans and animals in more than 98 countries across the globe placing more than 1 billion people at risk for the disease and killing more than 20,000 people per year. In the United States the disease is enzootic within the hunting dog population and vertical transmission has been identified as the primary route of transmission in this population. In Brazil the disease is endemic in the human population and enzootic in the dog population with vector and vertical transmission having been reported. In many diseases reports have found there is increased disease severity when an individual is co-infected with another organism. Case reports have suggested this may also occur with tick borne diseases and leishmaniosis in dogs but there is limited longitudinal data to support this relationship. Even less is known and understood regarding the risk factors and basic reproduction number, number of secondary cases one infected individual can cause in a susceptible population, of leishmaniosis in regards to vertical transmission. The goal of the work presented in this thesis is to address host factors related to the transmission of L. infantum and the way in which co-infections affect the progression of the disease both in the U.S. and in Brazil. Understanding the risk factors associated with the transmission of the parasite Leishmania infantum, the causative agent of the disease, are necessary to controlling and potentially elimination the disease. Utilizing a large prospective cohort and both active and passive surveillance it was identified that leishmaniosis can be maintained in a population via vertical transmission at prevalence rates similar to other endemic countries, 20%. With this knowledge an additional study examining a longitudinal cohort and assessing the impact of tick borne disease co-infections upon disease transmission was performed. It was identified that dogs exposed to three or more tick borne diseases were 11x more likely to progress to clinical disease (Adjusted RR: 11.64 95% CI: 1.22-110.99 p-value: 0.03) than dogs with no tick borne disease exposures. Furthermore, dogs with Leishmania and tick borne disease were 5x more likely to die within the study (RR: 4.85 95% CI: 1.65-14.24 p-value: 0.0051). When examining this relationship in a cross-sectional study in Brazil it was found that dogs with multiple tick borne disease exposures had 1.68x greater risk of being positive for Leishmania (Adjusted RR: 1.68 95% CI: 1.09-2.61 p-value: 0.019). Using a retrospective cohort of dogs and information regarding their dam’s diagnostic status near the time of pregnancy risk factors associated with vertical transmission and the basic reproduction number were calculated. It was found that dogs who were born to dams that were ever diagnostically positive for exposure and/or infection with L. infantum were 13.84x more likely become positive for L. infantum within their lifetime (RR: 13.84 95% CI: 3.54-54.20 p-value < 0.0001). The basic reproduction number for vertically transmitted L. infantum within this cohort was 4.16. The results of these studies suggest that leishmaniosis can be maintained in a population through vertical transmission. Furthermore, the studies show the risk factors associated with vertical transmission relate to the mother’s diagnostic status at time of pregnancy. The results of the co-infection studies highlight the importance of tick prevention in order to reduce disease progression. With increased disease severity associated with increased transmission to potential vectors these studies underline the need for immunotherapies and prevention measures to reduce disease progression in order to reduce transmission. Furthermore, these studies highlight the need for public health control and prevention programs to address vertical transmission if elimination of the disease is to ever be successful.

Thirty-three 1,4-dihydropyridine diastereomeric pairs were synthesized and the structure-activity relationship studied in a Plasmodium falciparum in vitro model. Twenty-nine of these derivatives contained a 6-position oxygen, with 2.31, 2.32, 2.52 and 2.53 having single and double digit nanomolar activities. This SAR study revealed some insightful information about the 1,4-dihydropyridine substitution pattern. Substitution at the 7-position other than 3,4-dimethoxy severely reduced the activity. 4-phenyl substitution with 2- or 4- halo or methyl formed active compounds while substitution at the 3-position or with methoxy or conjugated aryl systems resulted in inactive compounds. The 2-position was found to majorly affect the activity, with groups larger than methyl being the most active. The other four derivatives contained a 6-position methylene, with 2.1, 2.59 and 2.60 having single nanomolar activities. Lastly, stereochemistry was revealed to play an important role in the activity of 2.1. One stereoisomer, (+)-trans-2.1, had subnanomolar activity in two assays. Another stereoisomer, (4S,7S)-2.1, had nanomolar activity. The other two stereoisomers were inactive. A series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against Leishmania donovani and Leishmania amazonensis intracellular amastigotes. A structure-activity and structure-property relationship study was conducted in part using the Topliss operational scheme to identify new lead compounds. This study led to the identification of quinazolines with EC50s in the single digit micromolar or high nanomolar range in addition to favorable physicochemical properties. Quinazoline 3.23 also displayed efficacy in a murine model of visceral leishmaniasis, reducing liver parasitemia by 37% when given by the intraperitoneal route at 15 mg/kg/day for five consecutive days. Their antileishmanial efficacy, ease of synthesis, and favorable physicochemical properties make the N2,N4-disubstituted quinazoline-2,4-diamine compound series a suitable platform for future development of antileishmanial agents. A similar series of N2,N4-disubstituted quinazoline-2,4-diamines has been synthesized and tested against methicilin-resistant Staphylococcus aureus (MRSA) and multi-drug resistant strains of Acinetobacter baumannii. Quinazolines with MICs in the single digit micromolar or high nanomolar range were identified via SAR. In a murine model of MRSA infection, 1x the MIC for quinazoline 4.47 allowed for the survival of all tested mice at the end of a one week study. An in vivo model of A. baumannii was also undertaken using a Galleria mellonella model of infection. Quinazolines 4.74-4.76 afforded an increased protection of 87.5% when compared to the control experiments, with 70% of the wax worms surviving until day three. The observed potencies of frontrunner compounds in in vivo assays and their ease of synthesis make N2,N4-disubstituted quinazoline-2,4-diamines a suitable platform for the future development of anti-bacterial agents.

Dimorphic Leishmania donovani parasites exist as promastigotes in the sandfly vector and differentiate into amastigotes once injected into the skin of human hosts during a blood meal. The mechanisms and signals that are involved in triggering differentiation are not well understood in Leishmania. We have investigated whether tyrosine phosphorylation is a possible signalling component. Differential levels of tyrosine-phosphorylated proteins were observed in extracts from in vitro promastigote and amastigote cultures, with an overall reduction in the latter stage. Following this observation, the inhibition of tyrosine phosphorylation was examined in promastigotes using Tyrphostin AG1433, a broad-spectrum tyrosine phosphorylation inhibitor. AG1433 treated in vitro promastigote cultures differentiate into amastigote-like morphology, have reduced tyrosine phosphorylation level, and express the amastigote-specific marker A2 proteins. Our studies demonstrate that signal transduction mechanisms involving tyrosine phosphorylation/dephosphorylation events are involved in controlling L. donovani promastigote differentiation into amastigote forms.

This thesis describes the results obtained from the phytochemical analysis of Matricaria chamomilla, and the microbial transformation of oxymatrine (85) and harmine (87), as summarized below. 1. Chemical investigation of the crude methanolic extract of Matricaria chamomilla resulted in the isolation of a new natural product, matriisobenzofuran (72), along with four known compounds: apigenin (73), apigenin-7-O-β-glucopyranoside (74), scopoletin (75), and fraxidin (76). The structures of compounds 72-76 were elucidated with the aid of extensive NMR and mass spectroscopic studies. All of the aforementioned compounds showed moderate to good inhibitory activities against glutathione S-transferase, an enzyme which has been implicated in the resistance of cancer cells to chemotherapeutic agents. These compounds were also evaluated for antioxidant activity and displayed moderate to good free radical scavenging activity. Additionally, compounds 72-76 were screened for anti-leishmanial activity. Compounds 75 and 76 were significantly active in this assay, while the remaining compounds were weakly active. In the antibacterial and antifungal assays, compounds 72-76 were not active. 2. The second part of this thesis deals with the biotransformation studies on oxymatrine (85) and harmine (87). Oxymatrine (85) was metabolized to the deoxy analogue, matrine (84) by Penicillum chrysogeneum (ATCC 9480), Cunninghamella bainieri (ATCC 9244), Cunninghamella blakesleena (ATCC 9245 and 8688A), Curvularia lunata (ATCC 12017), and Fusarium sp. In the time-based analysis of this transformation, the metabolism of oxymatrine (85) could be detected after 48 hours of incubation. Additionally, incubation of harmine (87) with Mucor plumbeus (ATCC 4740) resulted in the isolation of harmine-N-oxide (94). The biotransformed products (84 and 94) were identified using IR, UV, NMR, and mass spectroscopic techniques. Compound 94 was evaluated for its ability to inhibit the enzyme acetylcholinestrase, whose overexpression has been linked to Alzheimer’s disease, and was found to possess weaker activity than harmine (87).

Leishmania are protozoan parasites that are transmitted by a sand fly vector. These parasites affect not only humans but also wild animals including domestic dogs and rodents, which form an additional challenge and public health problem to control the disease. Leishmaniasis is an important disease with worldwide distribution, including Saudi Arabia, the Middle East, and other tropical and subtropical areas around the world. Due to the expansion of irrigation and agricultural activities, more exposure to sand fly occurs, which leads to the expansion of leishmaniasis infections as newly emerging disease. Emerging drug resistance in leishmaniasis is an additional problem, contributed by enzymes involved in the detoxification of pharmacological agents and other xenobiotics. Cytochrome b5 reductase (Cb5r) has a high pharmacological significance because of its essential role in fatty acid elongation, biosynthesis of cholesterol (humans) or ergosterol (Leishmania, fungi), and cytochrome P450-mediated detoxification of xenobiotics. Leishmania Cb5r has seven different isoforms whereas human has only one. Cb5r-7 isoform in Leishmania has closest homology to the human Cb5r. The three specific aims of this thesis project are focusing on (i) cloning of the Cb5r-7 isoform from Leishmania mexicana, (ii) its purification as recombinant His-tagged protein from E.coli, and (iii) its functional characterization as potential pharmacological target against Leishmania.

This thesis describes the results obtained from the phytochemical analysis of Matricaria chamomilla, and the microbial transformation of oxymatrine (85) and harmine (87), as summarized below. 1. Chemical investigation of the crude methanolic extract of Matricaria chamomilla resulted in the isolation of a new natural product, matriisobenzofuran (72), along with four known compounds: apigenin (73), apigenin-7-O-β-glucopyranoside (74), scopoletin (75), and fraxidin (76). The structures of compounds 72-76 were elucidated with the aid of extensive NMR and mass spectroscopic studies. All of the aforementioned compounds showed moderate to good inhibitory activities against glutathione S-transferase, an enzyme which has been implicated in the resistance of cancer cells to chemotherapeutic agents. These compounds were also evaluated for antioxidant activity and displayed moderate to good free radical scavenging activity. Additionally, compounds 72-76 were screened for anti-leishmanial activity. Compounds 75 and 76 were significantly active in this assay, while the remaining compounds were weakly active. In the antibacterial and antifungal assays, compounds 72-76 were not active. 2. The second part of this thesis deals with the biotransformation studies on oxymatrine (85) and harmine (87). Oxymatrine (85) was metabolized to the deoxy analogue, matrine (84) by Penicillum chrysogeneum (ATCC 9480), Cunninghamella bainieri (ATCC 9244), Cunninghamella blakesleena (ATCC 9245 and 8688A), Curvularia lunata (ATCC 12017), and Fusarium sp. In the time-based analysis of this transformation, the metabolism of oxymatrine (85) could be detected after 48 hours of incubation. Additionally, incubation of harmine (87) with Mucor plumbeus (ATCC 4740) resulted in the isolation of harmine-N-oxide (94). The biotransformed products (84 and 94) were identified using IR, UV, NMR, and mass spectroscopic techniques. Compound 94 was evaluated for its ability to inhibit the enzyme acetylcholinestrase, whose overexpression has been linked to Alzheimer’s disease, and was found to possess weaker activity than harmine (87).

Made available in DSpace on 2014-08-13T14:50:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2011Bitstream added on 2014-08-13T18:01:18Z : No. of bitstreams: 1 ribeiro_es_me_araca.pdf: 554726 bytes, checksum: 120b6f528464317c0d52e748f3f81855 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A importância da leishmaniose visceral no contexto da saúde pública tem aumentado na ultima década. A manifestação da leishmaniose visceral nos cães é variável e estudos sobre a genética e a sua relação com a leishmaniose visceral têm indicado alguns genes envolvidos na susceptibilidade e resistência a esta doença. O objetivo deste estudo foi avaliar a relação entre as variantes alélicas dos gene Slc11a1 (solute carrier family 11 member 1) e DLA-DRB1 (dog leukocyte antigen) com a positividade para leishmaniose, determinada por meio da amplificação de DNA de cinetoplasto de Leishmania sp., por PCR, e pela presença de anticorpos anti-Leishmania sp. ao teste ELISA indireto. Foram observadas associações estatisticamente significantes entre a positividade para leishmaniose e: i. a presença do alelo (141, 145 ou 149) da região microssatélite (TAAA)n localizada no íntron 1 do gene Slc11a1 (P< 0,0001) ii. a variação do número de guaninas (8 ou 9 G) da região promotora (P=0,0280) (em ambos os casos quando analisados separadamente) e iii. a presença de qualquer dos alelos da região microssatélite associado a 8 ou 9 G da região promotora (P= 0,0025). A presença do alelo 141 foi estatisticamente associada à negatividade em cães (P<0,0001). Não foi observada associação estatisticamente significante entre a positividade à leishmaniose e a freqüência dos alelos do exon 2 do gene DLA- DRB1, embora tenha sido observada associação significante entre a freqüência dos alelos DRB1-W, DRB1*00101 ou DRB1-T e o resultado negativo para leishmaniose, podendo ser estes potenciais marcadores para resistência à leishmaniose em cães / The importance of visceral leishmaniasis in the context of public health has increased in the last decade. The manifestation of visceral leishmaniasis in dogs is variable and genetic studies and its relationship with visceral leishmaniasis have shown some candidate genes involved in susceptibility and resistance to this disease. This study aimed at evaluating the relationship between polymorphism of the genes Slc11a (solute carrier family 11 member 1), and DLA-DRB1 (dog leukocyte antigen) and the positivity to leishmaniasis determined by the amplification of Leishmania sp. kinetoplast DNA by PCR and the presence of anti-Leishmania sp. the indirect ELISA. We observed statistically significant associations between the leishmaniasis positive diagnosis and i. the presence of intron 1 microsatellite region alleles (141, 145 or 149) (P<0.0001), ii. the variation on the guanine number (8 or 9) in the promoter region (P=0.0280) (in both cases when analyzed separately), iii. the presence any of the microsatellite alleles associated to 8 or 9 G in the promoter region (P=0.0025). Microsatellite allele 141 was associated to negativity to leishmaniasis (P<0.0001). There was no association between the presence of any of the exon 2 DLA-DRB1 alleles and the leishmaniasis positive animals, although significant association between the frequency of the alleles DRB1-W, DRB1*00101 or DRB1-T and negative results to leishmaniasis were observed. These might be potential markers for dog’s resistance to visceral leishmaniasis

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-07Bitstream added on 2014-06-13T19:26:23Z : No. of bitstreams: 1 dossi_acs_me_jabo.pdf: 247848 bytes, checksum: 85bd25465d561a1526cea385be01c24d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O cão é o principal reservatório da Leishmania (Leishmania) chagasi, o parasita responsável pela Leishmaniose Visceral (LV) nas Américas. Na (LV) estudos em modelos experimentais, têm mostrado diferenças na resposta imunológica entre macho e fêmea. Na (L VC) tais estudos, não foram realizados. Este trabalho investigou em cães assintomáticos machos e fêmeas, naturalmente infectados por Leishmania (L.) chagasi, os níveis séricos de IgG contra antígenos totais de Leishmania, o nível de IL-10 e IFN-y, no sobrenadante do extrato do baço e fígado, e a citocina regulatória TGF-j3 no sobrenadante do extrato do baço e fígado, e sua produção natural no sobrenadante de cultura de células esplênicas. Os níveis de anticorpos anti-L. (L.) chagasi da classe IgG nos cães assintomáticos machos e fêmeas, não apresentaram diferença significativa entre o sexo. O nível de IL-10 mostrou-se elevado, no sobrenadante do extrato do fígado em cães infectados machos e fêmeas assintomáticos e apresentou dominância marcante em relação às outras citocinas. O nível do TGF-j3 mostrou-se aumentado no extrato do baço. O nível de IFN-y no baço dos grupos avaliados foi quantitativamente menor que as citocinas IL-10 e TGF-B. diferindo do observado no fígado onde foi observado uma baixa produção de TGF-j3 em relação ao IFN-y. No baço, o IFN-y apresentou diferença significativa em fêmeas assintomáticas quando comparadas com os machos assintomáticos. A observação da predominância das citocinas TGF-j3 no baço, e IL-10 no baço e fígado, em modelo ex vivo, sugere uma polarização da resposta imunológica para o padrão Th2 em cães naturalmente infectados por Leishmania (L.) chagasi. / The dog is the main reservoir of Leíshmanía (Leíshmanía) chagasí, the parasite responsible for Visceral Leishmaniasis (VL) in the Americas. Experimental studies on VL have shown differences in the immune response of males and females. Such studies have not been carried out on canine visceral leishmaniasis. This study investigated the seric levei of IgG against Leíshmanía total antigens, the levels of IL-10 and IFN-y in the supernatant of spleen and liver extracts, the regulatory cytokine TGF-131 in the supernatant of spleen and liver extracts, and its natural production in the supernatant of spleen cell culture, in male and female asymptomatic dogs naturally infected by Leíshmanía (L.) chagasi. The levels of anti-L. (L.) chagasí IgG antibodies in male and female asymptomatic dogs did not differ significantly between the sexes. IL-10 levei was high in the supernatant of the liver extract of male and female asymptomatic infected dogs and predominated over the other cytokines. TGF-131 levei was increased in the spleen extract. The levei of IFN-y in the spleen of the group evaluated was quantitatively lower than the IL-10 and TGF-131 cytokine levels, different from what was observed in the liver, where a low productíon of TGF-131 was observed when compared to IFN-y. In the spleen, IFN-y presented a significant difference in the asymptomatic females when compared to the asymptomatic males. The observation of a predominance of TGF-131 cytokines in the spleen and of IL-10 in the spleen and liver of an ex vívo model suggests a polarization of the immune response towards the Th2 pattern in dogs naturally infected by Leíshmanía (L.) chagasí.