Made available in DSpace on 2016-08-17T15:00:43Z (GMT). No. of bitstreams: 1 Raquel Silva Fonteles.pdf: 595737 bytes, checksum: 1cef131034266e95108a1695a3835c0b (MD5) Previous issue date: 2009-02-12 / In this study we examined, whether L. whitmani is susceptible to experimental infection by L. brasiliensis, causing Cutaneous and Mucocutaneous Leishmaniasis, and L. amazonensis, causing Diffuse Cutaneous Leishmaniasis. L. whitmani individuals were collected to establish a colony free from these parasites. Mice were experimentally infected with L. brasiliensis or L. amazonensis parasites. Female of this L. whitmani colony were permitted to feed on mice exhibiting clinical signs of the disease. Phlebotominae infected with parasites were detected by amplifying a Leishmania 120 bp specific kinetoblast DNA by means of PCR. The L. whitmani infection rate for Leishmania brasiliensis and Leishmania amazonensis was 65.2% and 47.4%, respectively and confirmed that the vector is susceptible to experimental L. brasiliensis and L. amazonensis infection. Comparison of the infection rates revealed statistically significant differences. The high L. whitmani infection rate together with epidemiological and entomological studies, verified that L. whitmani is responsible for L. brasiliensis infection. Remarkably, here we report for the first time that L. whitmani is vulnerable to experimental L. amazonensis infection and may be a vector of this pathogen and the distributor for Diffuse Cutaneous Leishmaniasis. / Este estudo teve como objetivo a análise da suscetibilidade experimental de Lutzomyia whitmani para infecção com Leishmania brasiliensis causadora da Leishmaniose Cutânea e Cutâneo-Mucosa e Leishmania amazonensis causadora da Leishmaniose Cutânea Difusa. Para isso, uma colônia de L. whitmani foi estabelecida com a finalidade de fornecer espécimes livres destes parasitas que foram submetidos à alimentação em camundongos experimentalmente infectados com L. brasiliensis e L. amazonensis. A infecção com parasita foi detectada a partir da amplificação por PCR de fragmentos de 120 pb de DNA cinetoplasto do gênero Leishmania sp. As taxas de infecção encontradas foram de 65,2% e 47,4% para L. brasiliensis e L. amazonensis, respectivamente, sendo, portanto, L. whitmani suscetível à infecção experimental por estas espécies de Leishmania. A comparação entre as taxas de infecção mostrou diferenças significativas. Assim, concluímos que a alta taxa de infecção desse vetor com L. brasiliensis associados à estudos epidemiológicos e entomológicos podem responsabilizá-lo pela transmissão da Leishmaniose Cutânea e Cutâneo-Mucosa. Notavelmente, pela primeira vez foi detectado que este flebotomíneo é suscetível à infecção experimental por L. amazonensis podendo ser um vetor para transmissão desse patógeno e dispersor da Leishmaniose Cutânea Difusa.

Nós investigamos os efeitos da saliva de Lutzomyia longipalpis capturados no campo e colonizados em laboratório, na evolução da lesão e imunomodulação da infecção por Leishmania (Leishmania) amazonensis, uma espécie que é endêmica na América do Sul, onde causa Leishmaniose Cutânea, Leishmaniose Cutânea Disseminada Bordeline e Leishmaniose Cutânea Difusa Anérgica, com conseqüências graves aos pacientes. Com o intuito de comparar o efeito dos dois tipos de extrato de glândula salivar, camundongos C57BL/6 foram inoculados subcutaneamente no coxim plantar das patas traseiras e nas orelhas com 106 formas promastigotas de Leishmania (L.) amazonensis na presença de extrato de glândula salivar de vetores de captura e de colônia. O tamanho da lesão foi significantemente menor nos camundongos infectados com extrato de glândula salivar de vetores capturados, o que também determinou uma infiltração menos proeminente de macrófagos nas lesões e uma resposta Th2 mais branda quando comparada com aqueles inoculados com extrato de glândula salivar de vetores colonizados. Recentemente, foi mostradas diferenças nos compostos protéicos das glândulas salivares que poderiam parcialmente justificar a expressão da lesão. Portanto, nossos achados ressaltam que a extrato de glândula salivar de flebótomos provavelmente não desempenha um papel importante na exacerbação da infecção por Leishmania já que a transmissão natural do parasito ocorre através de vetores selvagens e não através de vetores colonizados em laboratório / We investigated the effects of Lutzomyia longipalpis saliva of vectors captured in the field and colonized in laboratory on the lesion evolution and immunomodulation of the infection by Leishmania (Leishmania) amazonensis, a species that is endemic in South America where it causes cutaneous leishmaniasis, borderline disseminated cutaneous leishmaniasis and anergic diffuse cutaneous leishmaniasis, with devastating consequences to the patients. In order to compare the effect of both salivas, C57BL/6 mice were inoculated subcutaneously into the hind footpads or into the ear dermis with 106 promastigotes in the presence of salivary gland homogenate from wildcaught and lab-colonized vectors. Lesion size was significantly lower in the mice infected with saliva from wild-caught sandflies that also determined a less prominent infiltration of macrophages in the lesions and a weaker Th2 response in comparison with those co-inoculated with colonized saliva. Recently, differences were shown in the protein compounds in the salivary glands that could partially account for the lesion size outcome. In conclusion, our findings highlight that phlebotomine saliva probably does not play an important role in the exacerbation of Leishmania infection as the natural parasite transmission occurs through wild and not laboratory colonized vectors.

INTRODUÇÃO: A leishmaniose visceral (LV), pertence ao grupo de endemias consideradas prioritárias no mundo. É uma doença grave e com poucas opções de tratamento e, mesmo quando adequadamente tratada, possui um nível de letalidade de 5 a 7%. Apesar da expansão da doença, o tratamento para leishmaniose visceral não obteve avanços. O tratamento da leishmaniose possui apenas dois grupos de medicamentos utilizados: os antimonoiais e os não-antimoniais. Os compostos nitroheterocíclicos foram utilizados pois acredita-se que a eficácia desta classe de compostos está na habilidade desses compostos inibirem a tripanotiona redutase. Os compostos foram obtidos por via sintética e refinados pelo método QSAR por remodelagem molecular. Foram obtidos séries de compostos nitroheterocíclicos, onde a série BSF (derivados 5-nitro-2-furfurilideno - azometínicos) foi utilizada para avaliar a bioatividade in vitro destes compostos frente à Leishmania. Para avaliação da atividade anti-Leishmania foram usadas formas promastigotas de Leishmania (Leishmania) infantum. Os métodos utilizados para determinar a atividade anti-Leishmania dos compostos da série BSF foram os métodos colorimétrico 3-[4,5-dimetiltiazol-2-il] 2,5 brometo de difeniltetrazólio (MTT), apoptose por Anexina V e reação de Griess (dosagem de NO). Ao comparar os resultados com os respectivos controles positivos e com a Anfotericina B, foi observado que a atividade anti-Leishmania em formas promastigotas de L. L. infantum dos compostos foi menos significativa quando comparado com anfotericina B. Porém, ao comparar os resultados de macrófagos infectados e citotoxicidade com a anfotericina B, os compostos mostraram resultados expressivos, ao apresentarem baixa taxa de citotoxicidade e significativa diminuição da proliferação intracelular. A expressiva produção de nitrito pelos macrófagos infectados tratados com os compostos da série BSF sugere-se que estes compostos possuam uma ação indireta de potencialização de produção de NO nas células hospedeiras. Os resultados in vitro utilizando derivados nitroheterocíclicos mostraram atividade destes sobre formas promastigotas e amastigotas intracelulares, e são compostos em potencial para avaliação de efeito in vivo contra leishmaniose visceral / INTRODUCTION: Visceral leishmaniasis (VL) belongs to the group of endemics prioritized worldwide. It is a serious disease with few treatment options, and even when adequately treated, it has a lethality level of 5 to 7%. Despite the expansion of the disease, treatment for visceral leishmaniasis has not made progress. Treatment of leishmaniasis has used only two groups of drugs: antimonial and non-antimonial. Nitro-heterocyclic compounds were used in this study because it is believed that the effectiveness of this class of compounds is the ability to inhibit trypanothione reductase. The compounds were obtained synthetically and refined by QSAR method molecular remodeling. Nitro-heterocyclic series of compounds were obtained, where the BSF series (derived from 5-nitro-2-furfurylidene - azometínicos) was used to evaluate the in vitro bioactivity of these compounds against the Leishmania. To evaluate the anti-Leishmania activity, promastigotes of Leishmania (Leishmania) infantum were used. The methods used to determine the anti-Leishmania activity of the BSF series compounds were colorimetric methods 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide (MTT), apoptosis by Annexin V, and Griess reaction (IN dosage). By comparing the results with the respective positive controls and with amphotericin B, it was observed that the anti-Leishmania activity of L. L. infantum promastigotes of the compounds was less significant when compared with amphotericin B. However, comparing the results of infected macrophages and cytotoxicity with amphotericin B, compounds showed significant results, presenting low cytotoxicity rate and significant decrease in intracellular proliferation. The significant nitrite production by infected macrophages treated with the BSF series compounds suggested that these compounds have an indirect potentiation action in the NO production in the host cells. In vitro results using nitro-heterocyclic derivatives showed their activity on these promastigotes and intracellular amastigotes, and presented them as potential compounds for in vivo effect evaluation against visceral leishmaniasis

A Leishmaniose visceral é um problema que cresce no Estado de São Paulo afetando o homem e o cão. Os exoantígenos da membrana das leishmanias são liberados no meio de cultura. Os exoantígenos são importantes na indução da imunidade mediada pelas células T e B estimulando a produção elevada de anticorpos. Realizamos uma avaliação comparativa por ELISA e Immunoblotting de exoantígenos e antígenos totais de L. (L.) amazonensis, L. (V.) braziliensis e L. (L.) chagasi, no diagnóstico da leishmaniose visceral humana e canina. Obteve-se por ELISA sensibilidade de 100% para ambos os preparados antigênicos independente da espécie de Leishmania. A melhor especificidade em humanos e cães foi com os exoantígenos. O exoantígeno da L. (L.) chagasi teve a melhor especificidade e média de absorbâncias comparadas aos das outras espécies (p<0.005). Para o hospedeiro humano o ELISA com exoantígenos, não discriminou pacientes com leishmaniose cutânea e ou mucocutânea. O Immublotting dos exoantígenos de L. (L.) chagasi (IBleish) apresentou 100% de sensibilidade e especificidade para os cães. Os dados do IBleish-L. (L.) chagasi demonstraram a possibilidade de sua utilização como método confiável para a confirmação do diagnóstico da leishmaniose canina. / The visceral leishmaniasis is a new problem that grows in the State of São Paulo, affecting men and dogs. The exoantigens from the membrane of the Leishmania, are released out of culture medium. The exoantigens are important in the induction of immunity mediated by T and B cells, stimulating the production of antibodies. This work was carried out an evaluation of ELISA and Immunoblotting using comparatively exoantigens and total antigens of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) chagasi for the diagnosis of canine and human visceral leishmaniasis. ELISA sensitivity was 100% for all different antigens, independent of the species of Leishmania employed. The best specificity for both human and dogs were obtained with exoantigens. Exoantigen from L. (L.) chagasi was what showed the best specificity and mean absorbance compared to those of other species (p<0.05). The ELISA with exoantigens for the human host not discriminate individuals with leishmaniasis cutaneous or mucocutaneous. The Immunoblotting that used the exoantigens of L. (L.) chagasi (IBleish) showed 100% sensitivity and specificity for the dogs. The IBleish-L. (L.) chagasi showed the possibility of its use as a reliable method to confirm the diagnosis of canine leishmaniasis.

This thesis is focused on the bloodmeal digestion of phlebotomine sand flies and its effects on Leishmania development within their midguts. In the first part, we studied various parameters of bloodmeal digestion in four sand fly species differing in susceptibility to Leishmania donovani to evaluate the effects on vector competence. Both proven vectors of L. donovani (Phlebotomus orientalis and P. argentipes) showed lower trypsin activity and slower formation of the peritrophic matrix (PM) than refractory species (P. papatasi and Sergentomyia schwetzi). Remarkably, P. orientalis and P. argentipes strikingly differed from each other in a time course of bloodmeal digestion. Phlebotomus orientalis females showed very slow bloodmeal digestion with low peaks of proteolytic activities and defecated around day five post bloodmeal. In contrast, P. argentipes females digested faster with a very high peak of chymotrypsin activity, their PM was present for only a short time and defecation was finished by day three post bloodmeal. We presume that the period between the degradation of the PM and defecation (i.e. time frame when Leishmania bind to the midgut to avoid expulsion with bloodmeal remnants), is one of crucial parameters affecting the establishment of Leishmania in the sand flies. In both natural...

Sand fly saliva contains proteins and peptides that have an important role in bloodfeeding. Some of those proteins are antigenic and repeated sand fly bites result in a specific antibody response of the bitten host. Antigenic salivary proteins of Phlebotomus orientalis, main vector of visceral leishmaniasis in Sudan and Ethiopia, were identified using immunoblot with dog sera. The 5 most promising antigens were expressed in an E. coli bacterial system. Subsequently, these proteins were tested in ELISA with sera of domestic animals from Ethiopia naturally exposed to P. orientalis, and with sera of mice bitten experimentally by this sand fly species. Salivary gland homogenate (SGH) was used as the positive control. The best antigenic properties were detected in two recombinant proteins, Yellow-related protein PorSP24 and ParSP25-like protein PorSP65, especially in tests with sheep and dog sera. However, nonspecific binding of dog sera was also detected using both antigens. In addition, we proved that sera of mice repeatedly bitten by P. papatasi and Sergentomyia schwetzi do not crossreact with SGH and the tested recombinant proteins of P. orientalis. In a second part of this thesis we designed peptides representing epitopes recognized by specific anti-saliva antibodies. Two peptides were derived from...

6 Abstract Leishmaniasis is a neglected tropical disease, which belongs to the top health problems because it is endemic in 98 countries in Asia, Africa, the Americas and the Mediterranean region, and is gradually expanding to new areas, including Central Europe and USA. Clinical manifestations of leishmaniasis include a diverse range of forms, ranging from non-lethal cutaneous leishmaniasis to potentially lethal visceral leishmaniasis. Asymptomatic cases are known to exist in endemic areas. Different species of Leishmania induce distinct symptoms, but even the patients infected by the same species develop different symptoms and may respond differently to the treatment. Thus, one of the challenges is to explain the observed variability of leishmaniasis that cannot be attributed to the currently known factors. To find novel regulatory factors of the disease we tested molecules that were shown to play role in other infections and mapped loci controlling parasite load after L. major infection. We also determined genetic control of survival after infection with tick-borne encephalitis virus (TBEV) in order to establish whether there are common elements in response to L. major and TBEV. Interferon-induced GTPases (guanylate-binding proteins, GBPs) play an important role in inflammasome activation and mediate...

Determina el efecto fotodinámico de ftalocianina de aluminio tetrasulfonada clorada (PcAlClS4) en el tratamiento de leishmaniasis cutánea inducida en Mesocricetus auratus (hámster dorado) con Leishmania (L.) amazonensis. Dos grupos de hámsteres de 6 semanas fueron inoculados intradérmicamente en la zona sacro lumbar con cultivos de promastigotes; estos fueron monitoreados hasta por 7 meses. Luego de 56 días de inoculación aparecieron lesiones características de la enfermedad, sobre las cuales se les aplicó PcAlClS4 tanto en crema como en solución; luego fueron irradiados de inmediato con una lámpara LED en campo oscuro. Después de 12 semanas pos-tratamiento se evidenció la disminución de la zona afectada, ocurriendo sólo con la aplicación en solución. Asimismo, se observó la diseminación (metástasis) del parásito hacia otras partes del organismo, esto se evidenció a través de cortes histológicos de los tejidos tratados y fueron coloreados con hematoxilinaeosina, permitiendo observar la presencia de células plasmáticas y amastigotes del parásito en todas las muestras analizadas. Los resultados obtenidos en la presente investigación permiten reformular propuestas alternativas en el uso de PcAlClS4 como TFD y sus limitaciones en el tratamiento de la leishmaniasis cutánea. / Tesis

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