Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-06-21T19:30:11Z No. of bitstreams: 1 RaquelFonteles.pdf: 2324366 bytes, checksum: 7839768f7ff6e0756295b82f795ae2bf (MD5) / Made available in DSpace on 2017-06-21T19:30:11Z (GMT). No. of bitstreams: 1 RaquelFonteles.pdf: 2324366 bytes, checksum: 7839768f7ff6e0756295b82f795ae2bf (MD5) Previous issue date: 2016-06-24 / Leishmaniasis are a group of neglected infectious and parasitic diseases, with major repercussions at global and national level. In the State of Maranhao, both the American Cutaneous Leishmaniasis (LTA) and the American Visceral Leishmaniasis (LVA) are endemic and have great prominence at the national scene. Within the state territory, one of the leading regions is the Lençóis Maranhenses National Park - PNLM, whose surroundings have undergone environmental and ecological damage caused by man, resulting from direct and indirect tourism activities. As a result of this process every year are recorded cases, mostly LTA ones. Goals. Facing such reality, this study aimed: to define the epidemiological profile of the LTA between the years 2007 and 2013, taking into account demographic (age and gender), occupational and chronological features of the affected populations (reported cases); to demonstrate through a cartographic tool, a survey of the timeline distribution of cases of LTA; and an interactive map to determine the risk rate for acquisition of LT. It has been also proposed to study the transmission cycle of the disease, based on the identification of species of sandflies, their sources of blood and their Leishmania genus parasites, and additionally determining vector capacity of Lu. Whitmani toward Le. Braziliensis and Le. Amazonians. Methodology. The county of Barreirinhas was selected for this study because it is the main gateway to PNLM. In short, investigation of LTA cases was carried out in SINAN files, comprising all neighborhoods of the county (158), through the cycle 2007-2013, and sandflies collections were held in 62 of these communities. The species were identified taxonomically and females analyzed at the molecular level to research for natural infection by Leishmania, along with the animals that served as a food source. Results. 458 positive cases of LTA were reported between the years 2007 and 2013, predominantly in men from rural areas and farmers. The cases were concentrated in the south central area of the county, all year round. There was some geographic expansion with the emergence of new outbreaks, however, it was observed the persistence of the former areas of occurrence of the disease. As for the risk rates, villages Sítio Barra, Engenho and Manoelzinho were those who were positive for all variables. On the other side, the communities Quebra, Santa Rosa and Fumaça, presented less risk, since almost all the variables studied were negative for these locations. As for the sandflies, 9,853 specimens of 18 species have been collected, all present in extra-domicile (forest) and only 12 occurring in peri-domicile areas. The incidence was higher in areas surrounding dwellings (56.2%) than in extra-domicile environment (43.8%) and females predominated (51.4%) over males (48.6%). The dominant species were Lu. whitmani (53.83%), Lu. longipalpis (25.60%), Lu. Lenti (11.69%), Lu. evandroi (5.50%) and Lu. flaviscutellata (0.87%). Natural infections due to Le. infantum, were found in females captured in peri-domicile in five villages and extra-domicile in other two. The species Lu. longipalpis, vector of Le. Infantum, indicated a positivity rate of 3.7% for this species. While species not considered insect vector of this leishmania, such as Lu. Lenti and Lu. Whitmani, even so presented infection rates of 0.6% and 0.9%, respectively. Rates of natural infection due to Le. braziliensis, were detected in peri-domicile in two locations; while in Lu. Whitmani, considered the vector of this Leishmania species, rate of 0.3% was found. The rate of infection due to Le. amazonensis was 8% for Lu. Flaviscutelata, captured in peri-domicile in two villages and extra-domicile in just one. Lu. whitmani was able to get infected with Le. Braziliensis and Le. Amazonians. Concerning food source hosts, the following were identified: equines (9.0%), human (1.4%), canine (27.4%), rodent (3.3%), fowl (20.9%) and hogs (37.9%). Conclusion. All elements of the epidemiological chain of Leishmaniasis were present within the area of study, both in peri-domicile and extra-domicile areas, which explains the endemic state of the disease in the region. / Introdução. As leishmanioses constituem um grupo de doenças infectoparasitárias negligenciadas, com grande repercussão a nível mundial e nacional. No Estado do Maranhão tanto a Leishmaniose Tegumentar Americana (LTA) como a Leishmaniose Visceral Americana (LVA) são endêmicas e apresentam grande destaque no cenário nacional. Dentro do território maranhense, uma das regiões de destaque é a do Parque Nacional dos Lençóis Maranhenses – PNLM, cujo entorno, tem sofrido danos ambientais e ecológicos gerados pelo homem, em função das atividades diretas e indiretas do turismo. Como resultado desse processo todos os anos são registrados casos, principalmente de LTA. Objetivos. Diante desta realidade, este trabalho objetivou definir o perfil epidemiológico da LTA entre os anos de 2007 e 2013, levando em consideração os fatores demográficos (faixa etária e sexo), ocupacionais e temporais das populações acometidas (casos notificados); demonstrar através de uma ferramenta cartográfica, um mapa da distribuição espaço temporal dos casos de LTA e um mapa interativo para determinação da taxa de risco para aquisição da LT. Também se propôs estudar o ciclo de transmissão da doença, a partir da identificação das espécies de flebotomíneos, de suas fontes de sangue e de seus parasitas do gênero Leishmania, e determinar capacidade vetorial de Lu. whitmani para Le. Braziliensis e Le. Amazonenses. Metodologia. O município de Barreirinhas foi selecionado para esse estudo porque constitui a principal porta de entrada ao PNLM. Em suma, fez-se o inquérito dos casos da LTA nos arquivos do SINAN de todas as localides do município (158), no período de 2007-2013, e realizou-se coletas de flebotómineos em 62 destes povoados. As espécies foram identificadas taxonomicamente e as fêmeas analisadas a nível molecular para pesquisa de infecção natural por Leishmania e dos animais que serviram como fonte alimentar. Resultados. Foram notificados 458 casos positivos de LTA entre os anos de 2007 e 2013 predominando nos homens provenientes da zona rural e lavradores. Os casos estavam concentrados na região centrosul do município, ao longo dos anos. Houve certa expansão geográfica com o surgimento de novos focos, entretanto, foi observada a persistência das antigas áreas de ocorrência da doença. Quanto às taxas de risco, os povoados Barra do Sitio, Engenho e Manoelzinho foram os que apresentaram positividade para todas as variáveis estudadas. Já os povoados Quebra, Santa Rosa e Fumaça, ofereceram menor risco, uma vez que, quase todas as variáveis estudadas deram negativas para essas localidades. Quanto aos flebotomíneos, foram coletados 9.853 exemplares de 18 espécies, todas presentes no extradomicílio (mata) e apenas 12 ocorreram nos peridomicílios. A abundância foi maior no peridomicílio (56,2%) do que no extradomicílio (43,8%) e as fêmeas predominaram (51,4%) sobre os machos (48,6%). As espécies dominantes foram Lu. whitmani (53,83%), Lu. longipalpis (25,60%), Lu. lenti (11,69%), Lu. evandroi (5,50%) e Lu. flaviscutellata (0,87%). As infecções naturais por Le. infantum, foram encontradas em fêmeas capturadas nos peridomicílios de cinco povoados e no extradomicilio de outros dois. A espécie Lu. longipalpis, vetora de Le. infantum, mostrou uma taxa de positividade de 3,7% para esta espécie. Enquanto espécies não consideradas vetoras desta leishmânia, como Lu. lenti e Lu. whitmani mostraram taxas de infecção de 0,6% e 0,9%, respectivamente. A taxa de infecção natural por Le. braziliensis, foi detectada nos peridomicílios de duas localidades; para Lu. whitmani, considerada vetora desta espécie de Leishmania foi encontrada taxa de 0,3%. A taxa de infecção por Le. amazonensis foi de 8% para Lu. flaviscutelata obtidos nos peridomicilios de dois povoados e no extradomicilio de um apenas. Lu. whitmani foi capaz de se infectar com Le. Braziliensis e Le. Amazonenses. Para fonte alimentar foram identificados os seguintes hospedeiros: equino (9,0%), humano (1,4%), cão (27,4%), roedor (3,3%), galinha (20,9%) e porco (37,9%). Conclusão. Todos os elementos da cadeia epidemiológica das leishmanioses estavam presentes na área do presente estudo, tanto no peridomicílio como no extradomicílio, o que explica a situação endêmica da doença na região.

Leishmanioses são doenças causadas por protozoários do gênero Leishmania e se apresentam sob forma tegumentar ou visceral. No Brasil, a leishmaniose tegumentar americana (LTA) é causada, na sua maioria, por Leishmania (Viannia) braziliensis e conhecem-se principalmente as formas cutânea (LC), mucosa (LM) e disseminada (LD) da doença. Na LTA as formas clínicas tem sido atribuídas a diferenças na resposta imune do hospedeiro, mas recentemente vinculam-se também à variabilidade intraespecífica da L. (V.) braziliensis. Neste estudo avaliamos se haveria variabilidade biológica nos isolados de L. (V.) braziliensis, provenientes de pacientes com LC, LM e LD, principalmente em resposta a fator de crescimento insulina-símile-I (IGF-I). Os fatores de crescimento do hospedeiro tem sido alvo de estudos no desenvolvimento das leishmanioses, sendo IGF-I um deles. Havíamos demonstrado em estudos anteriores, utilizando Leishmania (Leishmania) amazonensis, que IGF-I induz proliferação, aumentando a atividade da arginase, com geração de poliaminas e diminuindo a síntese de óxido nítrico. No presente estudo analisamos o efeito de IGF-I em L.(V.) braziliensis, espécie prevalente no Brasil. Avaliamos inicialmente as características dos diferentes isolados enquanto promastigota e no prosseguimento enquanto amastigota em células de linhagem monocítica humana THP-1, com e sem estímulo de IGF-I. Nossos dados sugerem que há diferenças na atividade da arginase basal entre os isolados de L. (V.) braziliensis, sendo maior naqueles provenientes de pacientes com LM. IGF-I aumentou a atividade da arginase nos isolados de LC e LD, mas não de LM. Nos isolados em forma amastigota nas células de linhagem monocítica humana THP-1, o efeito de IGF-I foi de aumento do parasitismo nos isolados de LC e LM e de diminuição com os de LD. Nos isolados de LD a atividade da arginase basal foi menor que nos de LC. Por outro lado, a produção de óxido nítrico tendeu a ser maior em isolados de LD quando sob estímulo de IGF-I. Os dados sugerem que diferenças nas características biológicas dos parasitos podem contribuir na apresentação clínica dos casos da LTA. / Leishmaniasis are diseases caused by protozoa of the genus Leishmania that may manifest as cutaneous or visceral disease. In Brazil, American tegumentary leishmaniasis (ATL) is caused mostly by Leishmania (Viannia) braziliensis and cutaneous (CL), mucosal (ML) and disseminated (DL) forms of the disease are known.The diversity of clinical manifestations has been attributed to differences in the host immune response, but recently it has also been related to intraspecific variability of L. (V.) braziliensis. In the present study we evaluated whether there were biological variability in different isolates of L. (V.) braziliensis from patients with CL, ML, and DL, mainly in response to insulin-like growth factor-I (IGF-I). Growth factors of the host have been investigated in the development of leishmaniasis including IGF-I. In previous studies using Leishmania (Leishmania) amazonensis IGF-I was shown to induce proliferation, to increase the activity of arginase, generating polyamines and to decrease the synthesis of nitric oxide. In this study we analyzed the effect of IGF-I in L. (V.) braziliensis, a species prevalent in Brazil. Initially we evaluated the characteristics of individual isolates as promastigote and further as amastigote within human macrophage cell line THP-1 with and without IGF-I stimulation. Our data suggest that there are differences in the basal arginase activity amongst isolates of L. (V.) braziliensis, being higher in those from patients with ML. IGF-I increased the activity of arginase in the isolates of CL and DL, but not of ML. In isolates in the form of amastigotes within THP-1 cells, IGF-I induced the increase of parasitism of isolates from CL and ML, and decrease of those from DL. In isolates of DL the basal arginase activity was lower than in those of CL. Moreover, the production of nitric oxide tended to be higher with isolates of DL upon IGF-I stimulation. The data suggest that differences in the biological characteristics of parasites may contribute to the diversity of clinical presentation of ATL.

Leishmania (Leishmania) amazonenses está associada à leishmaniose cutânea difusa (DCL) e localizada (LCL). LCL se apresenta como uma lesão única com cura espontânea e DCL apresenta nódulos não ulcerativos que se espalham pela pele. Até o momento não se conhecem os fatores que influenciam na apresentação destas manifestações. A virulência do parasito tem sido associada com a capacidade de sobreviver no macrófago ativado. CD200 é uma glicoproteína reguladora que ativa o CD200R, gerando a inibição da resposta próinflamatória do macrófago. Recentemente foi demonstrado que no processo de infecção por L. (L.) amazonenses, há indução de CD200 no começo da infecção, aumentando a supervivência do parasito. O objetivo principal foi determinar a associação da expressão de CD200 na infecção de macrófagos com isolados de L. (L.) amazonenses associados à LCL ou DCL. Observamos que o aumento da expressão da proteína nos tempos avançados de infecção está relacionado com um aumento na infectividade dos isolados e um maior número de parasitos por célula infectada. / Leishmania (Leishmania) amazonenses is one of the etiologic agents of two forms of leishmaniasis: diffuse (DCL) and localized (LCL) cutaneous leishmaniasis. LCL is presents a unique ulcerated lesion and DCL multiple non-ulcerative nodules. Until now, it is not well known which factors influence the development of LCL or DCL. The parasites virulence has been associated with the ability to survive inside the activated macrophage. CD200 is a glycoprotein that interacts with CD200R inhibiting the proinflammatory response of the cell. Recently it was revealed during the infection process in macrophages L. (L.) amazonenses enhances CD200 expression in the early stages of infection resulting in the intracellular survival of amastigotes. The aim of this study was to determine the association between the induction of CD200 during infection with L. (L.) amazonenses isolates associated with LCL or DCL. An increase in CD200 expression was noteworthy in the late stages of infection, which was also related to a higher infectivity rate of L. (L.) amazonenses isolates.

De 12 casos de leishmaniose tegumentar (LT) em AIDS, em São Paulo, sete do levantamento retrospectivo (1990 a 2001) e cinco do prospectivo (2001 a 2004), com contato prévio com área endêmica para leishmanioses, 50% eram usuários de drogas injetáveis (1990-2001). Apresentavam média de linfócitos T CD4+ de 77 células/mm3, um era C2 e 11, C3 (classificação de HIV, segundo CDC) e 70% tinha sorologia positiva para leishmanioses. As manifestações de LT em mucosa e pele eram diversificadas: úlcera única ou lesões múltiplas e polimórficas ou disseminadas, incluindo comprometimento genital em 4 casos. Todos receberam tratamento específico para leishmaniose e 50%, HAART. 50% recidivaram e 50% foram a óbito no período, independentemente do uso do HAART / From 12 cases of tegumentary leishmaniasis (TL) in AIDS, from São Paulo, seven from retrospective (1990 a 2001) and five from prospective studies (2001 a 2004), with previous contact with endemic areas for leishmaniasis, 50% were endovenous drug users (1990-2001). Presenting mean 77 CD4+ T cells/mm3, one was C2 and 11 were C3 (HIV classification, according to CDC) and 70% had positive serology for leishmaniasis. Presentation of TL in the skin and mucosa were diversified: single or multiple ulcers and polymorphic or disseminated lesions, including lesions in genital area in 4 cases. All were treated with anti-leishmanial drugs and 50% with HAART. 50% presented relapse and 50% died during follow up period, independently of use of HAART

Leishmania (Leishmania) amazonenses está associada à leishmaniose cutânea difusa (DCL) e localizada (LCL). LCL se apresenta como uma lesão única com cura espontânea e DCL apresenta nódulos não ulcerativos que se espalham pela pele. Até o momento não se conhecem os fatores que influenciam na apresentação destas manifestações. A virulência do parasito tem sido associada com a capacidade de sobreviver no macrófago ativado. CD200 é uma glicoproteína reguladora que ativa o CD200R, gerando a inibição da resposta próinflamatória do macrófago. Recentemente foi demonstrado que no processo de infecção por L. (L.) amazonenses, há indução de CD200 no começo da infecção, aumentando a supervivência do parasito. O objetivo principal foi determinar a associação da expressão de CD200 na infecção de macrófagos com isolados de L. (L.) amazonenses associados à LCL ou DCL. Observamos que o aumento da expressão da proteína nos tempos avançados de infecção está relacionado com um aumento na infectividade dos isolados e um maior número de parasitos por célula infectada. / Leishmania (Leishmania) amazonenses is one of the etiologic agents of two forms of leishmaniasis: diffuse (DCL) and localized (LCL) cutaneous leishmaniasis. LCL is presents a unique ulcerated lesion and DCL multiple non-ulcerative nodules. Until now, it is not well known which factors influence the development of LCL or DCL. The parasites virulence has been associated with the ability to survive inside the activated macrophage. CD200 is a glycoprotein that interacts with CD200R inhibiting the proinflammatory response of the cell. Recently it was revealed during the infection process in macrophages L. (L.) amazonenses enhances CD200 expression in the early stages of infection resulting in the intracellular survival of amastigotes. The aim of this study was to determine the association between the induction of CD200 during infection with L. (L.) amazonenses isolates associated with LCL or DCL. An increase in CD200 expression was noteworthy in the late stages of infection, which was also related to a higher infectivity rate of L. (L.) amazonenses isolates.

A leishmaniose tegumentar (LT) é uma zoonose causada por protozoário do gênero Leishmania e transmitida pela picada de diferentes espécies de flebotomíneos infectados. Pode acometer o ser humano no momento em que este participa acidentalmente do ciclo silvestre de manutenção da doença, por meio de atividades que necessitem de sua entrada no ambiente de mata ou mesmo devido a existência de moradias próximas a borda da floresta ou até mesmo no interior da mesma. Atualmente há cada vez mais evidências da transmissão por consequência das modificações no ambiente causadas pelo ser humano, levando à migração de algumas espécies de Leishmania e de seus vetores para as imediações dos domicílios. A LT tem como principal agente etiológico a Leishmania (Viannia) braziliensis na Amazônia brasileira e esta é responsável por uma forma extremamente grave de leishmaniose anérgica. Este parasito caracteriza-se por apresentar espectro de manifestações clínicas e imunológicas que variam do pólo anérgico ao polo hiperérgico. O presente estudo faz-se necessário para avaliar: a adaptabilidade de populações humanas residentes em áreas endêmicas de LT; como elas procuram se ajustar ao ambiente no qual vivem; como a evolução destas populações humanas pode se refletir nos seus sistemas imunológicos e, consequentemente, nas formas, mais ou menos graves, de apresentação da LT em área hiperendêmica. Foram identificados 17647 flebotomíneos, sendo esses classificados em 86 espécies. Dessas, 14 espécies atendem a classificação de vetores de Leishmania sp. e outras 7 espécies são apontadas como potenciais vetores de Leishmania sp. O presente estudo comprova a existência da espécie Lutzomyia longipalpis, espécie vetor da leishmaniose visceral (LV), patologia na qual pode evoluir ao quadro de óbito senão ocorrer o devido tratamento. Este é o primeiro registro da espécie no estado do Acre e na porção sul-ocidental da Amazônia, e corrobora os atuais estudos que apontam a expansão territorial da ocorrência da LV no Brasil e com a hipótese de expansão até áreas Andinas. / Cutaneous leishmaniasis (CL) is a zoonosis caused by a protozoan of the genus Leishmania and transmitted by the bite of different species of infected sand flies. It may affect the human being at the time when it accidentally enters the wild cycle of the maintenance of the disease, through activities requiring its entry into the forest environment or even due to the existence of households near the edge of the forests or even within the same. Currently there is increasingly evidence of transmission due to changes in the environment caused by the human being, leading to the migration of some species of Leishmania and its vectors to the immediate vicinity of households. The CL has as the main agent etiological the Leishmania (Viannia) braziliensis in the Brazilian Amazon and this is responsible for an extremely serious form of leishmaniasis anergic. This parasite is characterized by presenting a spectrum of clinical and immunological manifestations ranging from the pole anergic to the pole hyperergic. The present study is necessary to evaluate: the adaptability of human populations resident in the endemic areas of CL; how they seek to adjust to the environment in which they live; how the evolution of these human populations can reflect on their immune systems and, consequently, in the more or less severe forms of presentation of the CL in hyperendemic area. 17647 phlebotomines were identified, being classified in 86 species. Among them, 14 species meet the classification of vectors of Leishmania sp. and 7 other species are pointed out as potential vectors of Leishmania sp. The present study proves the existence of the species Lutzomyia longipalpis, vector species of visceral leishmaniasis (VL), pathology in which it may evolve into the death frame, but due treatment. This is the first record of the species in the state of Acre and in the western portion of the Amazon, and corroborates the current studies that point the territorial expansion of the occurrence of LV in Brazil and with the chance of expansion to Andean areas.

Introdução: ao longo da história da leishmaniose tegumentar (LT), o comprometimento do tecido ósseo foi pouco estudado, principalmente na forma clínica difusa (LCD). Os estudos que abordaram o tema concentraram-se nas observações clínicas, estudos radiográficos e na visão paleopatológica, numa tentativa de explicar as lesões ósseas que alguns indivíduos com LT apresentavam. A literatura sobre infecção óssea cita como agente causador de osteomielite no homem, principalmente, bactérias e fungos. Objetivo: estudar as alterações ósseas na leishmaniose cutânea difusa (LCD), através da cintilografia óssea trifásica, radiografias e histopatológico. Métodos: o presente estudo analisa a associação entre a Leishmania (Leishmania) amazonensis e as alterações ósseas em oito pacientes com LCD, procedentes de diversos pólos de produção da doença (LT) no Estado do Maranhão. Foram realizados, nesta investigação, dois estudos: observacional em humanos, utilizando exames de imagens (cintilografia óssea trifásica e radiografias) e histopatológico do tecido ósseo (Hematoxilina- Eosina, Imunohistoquímica, Ziehl-Neelsen e Gomori-Grocott); e um estudo experimental de infecção óssea crônica produzida pela Leishmania, em camundongos BALB/c, através do histopatológico (Hematoxilina-Eosina). Resultados: a análise clínica mostrou a presença de deformidades nas extremidades distais dos membros superiores e inferiores em 50% dos casos, com limitação funcional associadas às lesões cutâneas. Nos estudos de imagens, a cintilografia mostrou hipercaptação na fase tardia em várias áreas de uma mesma região, caracterizando processos inflamatórios múltiplos no tecido ósseo em todos os pacientes. A radiografia demonstrou características de reabsorção óssea parcial das falanges distais dos dedos das mãos e pés, bilateral em seis pacientes. Revelou, também, lesões líticas nas falanges proximais das mãos em dois pacientes. O exame histopatológico demonstrou a presença de um quadro de osteomielite crônica em quatro pacientes, sendo detectado a Leishmania. Foi realizada pesquisa para micobactérias e fungos no tecido ósseo, que foram negativas. O modelo experimental caracterizou um quadro de infecção óssea crônica, demonstrando o comprometimento do tecido cartilaginoso secundariamente. Conclusões: todos os pacientes apresentaram algum tipo de comprometimento ósseo. As deformidades localizaram-se nas extremidades distais em seis pacientes. A cintilografia óssea e a radiografia foram importantes na localização e no seguimento das lesões ósseas. Através do exame histopatológico ficou evidente a relação entre a L.(L.) amazonensis e infecção óssea, caracterizando um quadro de osteomielite crônica. Portanto, fica a proposta de modificação da classificação etiológica da osteomiielite crônica, incluindo o protozoário, Leishmania (L) amazonensis , como agente causador / Introduction: Since the early history of Cutaneous Leishmaniasis (CL), there are few studies on the bone involvement in CL, mainly related to the Cutaneous Diffuse Leishmaniasis (CDL). The studies were mainly related to the clinical observation, radiographical aspects and the paleopathological view, on the attempt to explain the bone lesions found in some individuals with CL. The literature on bone infection reports the bacteria and fungi as agents of osteomyelitis in man. Objective: to study the bone changes in Cutaneous Diffuse Leishmaniasis (CDL), through the scintilography, radiography and histopatological aspects. Methods: the study analyzed the association of the Leishmania (Leishmania) amazonensis and the bone changes in eight patients with CDL, from eight endemic poles of the disease in the State of Maranhão. Two studies were carried out: human observations using images exams such as scintilography, radiography and histopathology of the bone tissue using the Hematoxilin - Eosine, Ziehl Neelsen and the Gomori-Grocott stain and also by immunohistochemistry. An experimental study of chronic bone infection induced by the Leishmania in BALB/c mice, were carried out analyzed through the histopathology (Hematoxylin-Eosin). Results: the clinical analysis showed deformity in the upper and inferior extremities in 50% of the cases, with functional limitation. On the studies of images, the scintilography showed hipercaptation on the late phase, in different areas of the same region, characterizing multiples inflammation in the bone of all patients. The radiography demonstrated characteristics of bone partial reabsortion of the feet and hands, bilateral in six patients. Lithic changes were also detected in fingers, of two patients. The histopathology showed chronic osteomyelitis in four patients, with detection of the Leishmania ( L.) amazonensis parasite. The research for mycobacteria and fungi in the bone tissue were negatives. The experimental model showed chronic infection of the bone tissue, demonstrating that the cartilage was secondarily involved. Conclusion: all patients presented some type of bone changes. The deformities were located in the extremities in six patients. The scintilography of the bone and radiography were important for localization and follow up of the bone lesions. The histopathological exams showed the relationship between Leishmania (L) amazonensis and bone infection, characterizing chronic osteomyetitis. We proposed that the Leishmania (L) amazonensis should be included as agent of chronic osteomyelitis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Nas Américas, a leishmaniose visceral (LV) experimenta um processo de urbanização e o cão doméstico é considerado o principal reservatório da doença neste cenário, embora seu papel no ciclo de transmissão não esteja totalmente explicado. Este estudo teve como objetivo investigar, por meio da análise de dados espaciais e imagens de sensoriamento remoto, a relação de fatores ambientais com a ocorrência de infecção canina por Leishmania chagasi e sua correlação espacial com a doença humana na cidade de Teresina (Piauí - Brasil), onde foi relatada a primeira epidemia urbana de LV no Brasil. Os resultados são apresentados na forma de dois manuscritos, nos quais são utilizados dados georreferenciados obtidos por meio de um inquérito sorológico canino realizado durante o ano de 2011, em diferentes bairros com transmissão moderada ou intensa. No primeiro, a regressão logística multinível foi utilizada para correlacionar a prevalência da infecção canina com variáveis ambientais de quadrículas de 900m2 (30mx30m) onde os domicílios estavam localizados, ajustando para as características individuais dos cães (sexo, idade e raça) e da residência. Participaram desta análise 717 cães distribuídos em 494 domicílios e 396 quadrículas. Um percentual >16,5% da área da quadrícula coberta por pavimentação clara (ruas de terra ou asfalto antigo) foi a única variável ambiental associada com a infecção canina por L. chagasi (Odds ratio [OR] = 2,00, intervalo de 95% de confiança [IC95%]: 1,22 - 3,26). Estas áreas provavelmente correspondem àquelas mais pobres e com pior infraestrutura urbana, sugerindo a ocorrência de um padrão de transmissão intra-urbano similar aos padrões rurais e peri-urbanos da LV. No segundo manuscrito, a partir da análise hierárquica do vizinho mais próximo foi verificada a presença de sete clusters de maior concentração de cães soropositivos em relação aos soro negativos em áreas menos urbanizadas e com vegetação pouco densa. Participaram desta análise 322 cães distribuídos em cinco bairros. A relação espacial entre os caninos soropositivos e os casos humanos foi investigada através do método da distância média entre os pontos e analisada por meio do teste t. Foi encontrada uma maior proximidade de casos humanos em relação a cães soropositivos quando comparada à distância em relação aos soro negativos, sugerindo a existência de uma relação espacial entre a LV humana e a soropositividade canina. Os resultados contribuem para uma maior compreensão sobre a dinâmica da doença em meio urbano além de fornecer informações úteis para a prevenção e controle da LV em seres humanos. / In the Americas, visceral leishmaniasis (VL) undergoes an active process of urbanization and the domestic dog is considered to be the main reservoir of the disease in this scenario, although its role in the transmission cycle is not fully explained. This study aimed to investigate, by spatial data analysis and remote sensing images, the association between environmental factors with the occurrence of canine infection by Leishmania chagasi and their spatial correlation with the human disease in the city of Teresina (Piauí-Brazil), where first urban epidemic of LV in Brazil was reported. The results are presented in the form of two manuscripts, which used georeferenced data obtained through a serological canine survey carried out during 2011 canine, in localities with moderate or severe transmission. In the first manuscript, the multilevel logistic regression approach was used to correlate the prevalence of canine infection with the environmental variables of grids with 900m2 (30mx30m) where the residence was located, adjusting to the dogs' individual characteristics (gender, age and race) and characteristics of the residences. The environmental analysis was performed with 717 dogs distributed in 494 households and 396 grids. The percentage of the area of grid covered by clear pavement (bare soil or old asphalted streets) >16.5% was the only environmental variable associated with canine infection by L. chagasi (Odds ratio [OR] = 2.00, 95% confidence interval [95% CI]: 1.22 - 3.26). These probably correspond to impoverished areas with inadequate infrastructure suggesting the occurrence of an urban cycle similar to those observed in rural and periurban areas. In the second manuscript, by the hierarchical analysis of nearest neighbor, were checked seven clusters of highest concentration of seropositve dogs in relation to seronegative in less urbanized areas with sparse vegetation. This analysis was performed with 322 dogs distributed in five boroughs. The spatial relationship between seropositive dogs and human cases was investigated by the method of average distance between points and was analyzed by t-test. The human cases were closer to the seropositive dogs when compared to the distance from the seronegative ones, suggesting the existence of a spatial relationship between the human LV and canine infection. The results contribute to a greater understanding of the dynamics of urban disease and also provide useful information for the prevention and control of visceral leishmaniasis in humans.

De 12 casos de leishmaniose tegumentar (LT) em AIDS, em São Paulo, sete do levantamento retrospectivo (1990 a 2001) e cinco do prospectivo (2001 a 2004), com contato prévio com área endêmica para leishmanioses, 50% eram usuários de drogas injetáveis (1990-2001). Apresentavam média de linfócitos T CD4+ de 77 células/mm3, um era C2 e 11, C3 (classificação de HIV, segundo CDC) e 70% tinha sorologia positiva para leishmanioses. As manifestações de LT em mucosa e pele eram diversificadas: úlcera única ou lesões múltiplas e polimórficas ou disseminadas, incluindo comprometimento genital em 4 casos. Todos receberam tratamento específico para leishmaniose e 50%, HAART. 50% recidivaram e 50% foram a óbito no período, independentemente do uso do HAART / From 12 cases of tegumentary leishmaniasis (TL) in AIDS, from São Paulo, seven from retrospective (1990 a 2001) and five from prospective studies (2001 a 2004), with previous contact with endemic areas for leishmaniasis, 50% were endovenous drug users (1990-2001). Presenting mean 77 CD4+ T cells/mm3, one was C2 and 11 were C3 (HIV classification, according to CDC) and 70% had positive serology for leishmaniasis. Presentation of TL in the skin and mucosa were diversified: single or multiple ulcers and polymorphic or disseminated lesions, including lesions in genital area in 4 cases. All were treated with anti-leishmanial drugs and 50% with HAART. 50% presented relapse and 50% died during follow up period, independently of use of HAART

Μέχρι σήμερα δεν υπάρχει εμβόλιο για καμιά μορφή των λεϊσμανιάσεων και τα φάρμακα που χρησιμοποιούνται δεν είναι ασφαλή, αποτελεσματικά και οικονομικά προσιτά. Υπάρχει άφθονη επιστημονική πληροφορία για τα αντιλεϊσμανιακά φάρμακα που χρησιμοποιούνται με αναγνωρισμένα μειονεκτήματα όπως η τοξικότητα, η ανάπτυξη αντοχής, η υποχρεωτική παραμονή στο νοσοκομείο και το υψηλό κόστος. Επιπλέον η συλλοίμωξη με τον ιό του AIDS (HIV/VL) έχει σοβαρές επιπτώσεις στην επιδημιολογία, τη διάγνωση και την πρόγνωση της νόσου. Είναι προφανές ότι η εκδήλωση της σπλαγχνικής λεϊσμανίασης είναι “ευκαιριακή” όπως αποδεικνύεται από τα στοιχεία του Π.Ο.Υ., σύμφωνα με τα οποία οι ασθενείς με AIDS έχουν 3.000 φορές υψηλότερη συχνότητα VL σε σύγκριση με τον γενικό πληθυσμό. Για τα διάφορα είδη του γένους Leishmania υπάρχουν αξιόλογα ευρήματα από παρασιτολογικές, βιοχημικές, μοριακές και ανοσολογικές μελέτες, όμως τα θεραπευτικά πρωτόκολλα ενάντια των διαφορετικών κλινικών μορφών δεν είναι ικανοποιητικά. Γεγονός αποτελεί η αδήριτη ανάγκη για την ανάπτυξη νέων θεραπευτικών προσεγγίσεων κατά των λεϊσμανιάσεων που μαστίζουν έως και 1,7 εκατομμύρια ανθρώπους σε όλη την γη. Σήμερα το ενδιαφέρον για φυσικά προϊόντα βρίσκεται σε επικαιρότητα κυρίως με την αναζήτηση νέων χημικών ενώσεων αλλά και την επανεξέταση παλαιοτέρων σε σύγχρονα πλέον πειραματικά πρωτόκολλα. Μεταξύ αυτών των προϊόντων η ελαιοευρωπεΐνη, που προέρχεται από το δένδρο της ελιάς (Olea europaea), χαρακτηρίζεται από αντιοξειδωτική, αντιμικροβιακή και αντιφλεγμονώδη δράση. Η βιοφαινόλη αυτή φαίνεται να συμβάλλει επίσης στην μακροζωία αλλά και όταν χορηγείται σε πειραματόζωα με καρκινικούς όγκους είναι ικανή στο να τους συρρικνώνει ή ακόμη και να τους εξαφανίζει. Ο σκοπός αυτής της διατριβής είναι η διερεύνηση φυσικών προϊόντων προερχόμενων από τα φύλλα και τους καρπούς του δέντρου της ελιάς αλλά και από πάρα-προϊόντα του όπως τα υγρά απόβλητα ελαιοτριβείου. Επίσης εξετάσθηκαν δύο αδρά εκχυλίσματα από τον ερυθρό οίνο ποικιλίας ξινόμαυρο. Αμφότερες οι πηγές των εκχυλισμάτων αποτελούν θεμελιώδεις λίθους της Μεσογειακής διατροφής και οι βιβλιογραφικές αναφορές τα καθιστούν ως υποσχόμενους αντιλεϊσμανιακούς παράγοντες. Τα αποτελέσματά έδειξαν ότι τα δύο καθαρά φυσικά προϊόντα που εξετάσθηκαν, η υδροξυτυροσόλη και η ελαιοευρωπεΐνη επέδειξαν επιλεκτική και σημαντική αντιλεϊσμανιακή δραστικότητα εναντίον τριών ειδών πρωτοζώων του γένους Leishmania και συγκεκριμένα των L. infantum, L. donovani και L. major. Η διατριβή εστιάστηκε στη ελαιοευρωπεΐνη και αναδείχθηκε η ικανότητά της να προκαλεί ρυθμιζόμενο κυτταρικό θάνατο αποδεικνυόμενο από μορφολογικές αλλαγές σε προμαστιγότες L. donovani, λογαριθμικής φάσης ανάπτυξης, με τη χρήση συνεστιακής μικροσκοπίας. Η στρογγυλοποίηση των προμαστιγοτών, η συμπύκνωση της χρωματίνης καθώς και η έκθεση της φωσφατιδυλοσερίνης στην εξωτερική επιφάνεια της κυτταροπλασματικής μεμβράνης με τη χρήση κυτταρομετρίας ροής είναι φαινοτυπικά χαρακτηριστικά της επαγωγής ρυθμιζόμενου παρασιτικού θανάτου που αποδείχθηκε ότι ο μηχανισμός πρόκλησής του είναι ανεξάρτητος της παραγωγής ROS. Επιπλέον η ελαιοευρωπεΐνη προκάλεσε μια καθυστέρηση στο φυσιολογικό κυτταρικό κύκλο του παρασίτου οδηγώντας στον κατακερματισμό του DNA. Η αντιλεϊσμανιακή δράση της ελαιοευρωπεΐνης αποδείχτηκε και εναντίον των αμαστιγωτικών μορφών του είδους L. donovani που είχαν in vitro παρασιτήσει σε μακροφάγα της κυτταρικής σειράς J774Α.1. Αυτή η δράση συνοδεύτηκε από μία ενδομακροφαγική αύξηση ROS ενώ η in vitro χορήγηση ελαιοευρωπεΐνης σε μυελοειδή DCs αύξησε τον πληθυσμό που παράγει IL-12 δίχως να προκαλεί παράλληλη ωρίμανσή τους. Τα ανωτέρω οδήγησαν στη μελέτη της δράσης της ελαιοευρωπεΐνης σε in vivo πειραματικό μοντέλο σπλαχνικής λεϊσμανίασης. Σε L. donovani μολυσμένα BALB/c ποντίκια χορηγήθηκε ενδοπεριτοναϊκώς ελαιοευρωπεΐνη κάθε δεύτερη ημέρα για 28 συνεχόμενες ημέρες σε τρεις διαφορετικές δόσεις (45, 15 και 5mg/kg σωματικού βάρους). Βρέθηκε ότι και οι τρεις δόσεις περιόρισαν το παρασιτικό φορτίο του σπλήνα και του ήπατος στις 3 ημέρες και στις 6 εβδομάδες μετά το πέρας της χορήγησης της ελαιοευρωπεΐνης. Διαπιστώθηκε ότι τα πειραματόζωα που έλαβαν ελαιοευρωπεΐνη και περιόρισαν την ενδοκυτταρική εξάπλωση του παρασίτου στο σπλήνα και στο ήπαρ ανέπτυξαν μια ενισχυμένη ΤΗ1 τύπου ανοσολογική απόκριση με χαρακτηριστικό ισοτυπικό προφίλ αντισωμάτων IgG2α/IgG1 και μεταγραφικών παραγόντων Tbx21/GATA-3. Η υπερ-έκφραση των IL-12p40, IFNγ, TNFα και iNOS επιβεβαιώνει την πόλωση προς τον ΤΗ1 υποτύπο ανοσολογικής απόκρισης, ενώ η έκφραση των κυτταροκινών ρυθμιστές της TΗ2 ανοσολογικής απάντησης δεν είχαν καμία αυξητική τάση. Αυτό το φαινόμενο που προέρχεται αρχικώς από την ικανότητα της ελαιοευρωπεΐνης να διατηρεί χαμηλά τα επίπεδα της προφλεγμονώδους κυτταροκίνης IL-1β, επιτρέπει τον ανοσολογικό μηχανισμό του ξενιστή να ενεργοποιηθεί (μεταγραφή του Tbx21) αποτρέποντας την είσοδο του ξενιστή σε κατάσταση χρόνιας φλεγμονικής διεργασίας που ευνοεί τον παρασιτικό πολλαπλασιασμό. Απεναντίας ενεργοποιούνται μηχανισμοί παραγωγής των μικροβιοκτόνων μορίων ROS και NO• στο σπλήνα και στο ήπαρ. Η παραγωγή των ROS σε σπληνοκύτταρα ποντικών μολυσμένων με L. donovani ήταν δοσοεξαρτώμενη ενώ αντιθέτως η παραγωγή των ΝΟ• ήταν μη-δοσοεξαρτώμενη τόσο σε κύτταρα του σπλήνα όσο και του ήπατος. Αυτή η παραγωγή των ΝΟ• φαίνεται να επιδρά στην επιβίωση των παρασίτων χωρίς να αλλοιώνει τη φυσιολογική λειτουργία των σπληνοκυττάρων και των ηπατοκυττάρων εφ’ όσον τα μόρια γλουταθειόνης στα κύτταρα του ξενιστή ήταν επαρκή για να συντηρούν τη συνεχή επιβίωση του κυττάρου ξενιστή όπως παρατηρήθηκε με την μη-ενεργοποίηση του NF-kB. Επίσης η ελαιοευρωπεΐνη επιλεκτικά ρυθμίζει την γονιδιακή έκφραση των ενζύμων που σχηματίζουν τη γλουταθειόνη και την τρυπανοθειόνη εις βάρος των αντιοξειδωτικών αμυντικών μηχανισμών του παρασίτου. Συμπερασματικά, το σύνολο των αποτελεσμάτων που περιεγράφηκαν στη διδακτορική αυτή διατριβή, προτείνουν ότι η ελαιοευρωπεΐνη δρα ως ένα αντιλεϊσμανιακό φάρμακο με ανοσοτροποποιητικές ιδιότητες. Η διερεύνηση της συνδυαστικής δράσης κλασσικών αντιλεϊσμανιακών φαρμάκων με ασφαλή, μη-τοξικά και “φθηνά” φυσικά προϊόντα όπως η ελαιοευρωπεΐνη, παραμένει μια υποσχόμενη μελλοντική προοπτική. Επιπροσθέτως, ενδιαφέρουσα θα ήταν η διερεύνηση της δράσης της ελαιοευρωπεΐνης στην έκφραση έτερων γονιδίων που κωδικοποιούν αντιοξειδωτικά ένζυμα καθώς και στους κυτταρικούς πληθυσμούς που εμπλέκονται επίσης σε in vivo πειραματικό μοντέλο σπλαχνικής λεϊσμανίασης όπως τα δενδριτικά κύτταρα, τα κύτταρα φυσικοί φονιάδες και τα CD8+. Τέλος η ελαιοευρωπεΐνη μπορεί να δοκιμασθεί σε HIV/L. donovani πειραματικό πρωτόκολλο αφού η ελαιοευρωπεΐνη είναι ικανή στο να σταματάει τον πολλαπλασιασμό του ιού HIV. / Up to now there has been neither a vaccine against any form of leishmaniasis found nor drugs that are safe, effective and inexpensive. Plethora of recent data has showed that the existing antileishmanial drugs have numerous disadvantages such as toxicity, development of resistance, long hospitalization and high cost. In addition, HIV/VL coinfection has important epidemiological, clinical, diagnostic and prognostic implications. The two diseases are mutually reinforcing. Clearly the opportunistic manifestation of VL is demonstrated by the WHO records showing that 50-75% new VL cases in South Europe concern HIV patients that bring the incidences of VL 3,000 times more frequent than in the general population. Despite the advances in the parasitological, biochemical, molecular and immunological research using various Leishmania species, the treatment protocols applied against the different forms of the disease are not satisfactory. There is an unconquerable need for the development of new therapeutic treatments to combat leishmaniasis. Nowadays, the interest concerning plant natural products is certainly undergoing a renaissance. The remarkable chemical diversity present in natural products and the accumulated research data produced so far, demonstrate their significant effectiveness against parasitic diseases suggesting the hypothesis to be tested also against leishmaniasis. This was particularly evident in the case of natural products of the olive tree (Olea europaea). Among them oleuropein has most of olive oil’s antioxidant, anti-inflammatory, and disease-fighting characteristics and it is the biophenol that provides life extending benefits. In addition to its antimicrobial and antioxidant activity, when oleuropein was given to animals with tumors, the tumors completely regressed or in some cases even disappeared. The aim of this thesis is to investigate natural products obtained from the olive tree leaves, products and their by-products (olive mill waste waters) and red wine (xinomavro variety), major cornerstones of the Mediterranean diet, as natural sources for promising antileishmanial drugs in accordance with the considerations outlined above. Our results showed that both pure constituents, hydroxytyrosol and oleuropein, exhibited the highest and the most selectively significant leishmanicidal activity against parasites of three Leishmania species, L. infantum, L. donovani and L. major, among the natural products tested. We focused on oleuropein and we discovered that it was capable of causing substantial morphological alterations upon L. donovani logarithmic promastigotes depicted with confocal microscopy namely, cell rounding up, cellular volume reduction and chromatin condensation that were accompanied with phosphatidylserine exposure to the outer leaflet of the plasma membrane. Moreover, oleuropein was found responsible for a delay on normal L. donovani cell cycle progression which was extended up to DNA fragmentation. This regulated parasitic cell death induced by oleuropein appeared to be ROS-independent. Oleuropein’s leishmanicidal capacity was also evident against the amastigote form of the parasite using an in vitro experimental model based on J774.A1 macrophages infected with L. donovani. This activity is accompanied by an increase of inracellular ROS production and the ability of oleuropein to increase the percentage of IL-12 producing myeloid DCs when is administrated in vitro. This data suggested testing the leishmanicidal activity of oleuropein upon in vivo murine visceral model. Intraperitoneal administration of oleuropein every other day for 28 days in L. donovani infected BALB/c mice, led to the dramatic reduction of the parasite load in spleen and liver. Interestingly, oleuropein treated L. donovani infected BALB/c mice, switch towards a TH1 type of immune response characterized by relevant IgG2a/IgG1 isotype and Tbx21/GATA-3 transcription factor ratio. Analysis of the whole spleen tissue revealed small but significant fold changes in gene expression of TH1 versus TH2 type of cytokines between treated and non-treated L. donovani infected BALB/c mice. Particularly, the dominance of TH1 type of response was confirmed by the up regulation of IL-12p40, IFN-γ and TNF-α which triggers elevated expression of iNOS in splenocytes in oleuropein treated L. donovani infected BALB/c mice. Despite the presence of TH2 type of response, the abiding down regulation of IL-1β permits Tbx21 expression and prevents prolonged inflammatory process and thus to disease exacerbation. We also found that oleuropein promoted a dose-dependent ROS production on spleen cells from BALB/c mice infected with L. donovani while it was able to evoke a dose-independent NO production on spleen and liver cells. NO production seems to affect parasitic viability but did not alter physiological function of spleen and liver cells since glutathione cell reservoir is capable of maintaining the continuity of host cell survival confirmed by the absence of NF-kB2 expression increase. Furthermore, oleuropein selectively regulates the transcription of glutathione and trypanothione gene which encode their synthesis enzymes at expense of parasitic antioxidant defense mechanism. The overall conclusion of the research described in this thesis suggests that oleuropein acts as an effective antileishmanial drug with demonstrable immunostimulating properties and selectively induces parasitic death by microbicidal molecules. The option of testing the combining effects of classical antileishmanial drugs with safe, non-toxic and cost effective natural products like oleuropein remains a promising future perspective. Moreover, interesting remains the effect of oleuropein upon other antioxidant genes and cell populations that play crucial roles in the in vivo experimental visceral leishmaniasis such as mDCs, NK and CD8+ cells. Finally, oleuropein could be used upon HIV/L. donovani experimental protocol since oleuropein is also capable to cease viral multiplication.