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Scorza, Breanna M. "Interaction of human keratinocytes with Leishmania spp.: a comparative study of Leishmania infantum and Leishmania major". Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5847.
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Leishmaniasis refers to the group of diseases caused by pathogenic protozoan parasites of the genus Leishmania. Nearly all human Leishmania spp. infections are initiated in mammalian skin through the bite of the phlebotomine sand fly vector. However, clinical manifestations vary greatly with infecting species. Leishmania major establish infection locally within the skin and cause chronic ulcerating skin lesions at the local cutaneous site of inoculation, in a syndrome known as Cutaneous Leishmaniasis (CL) Leishmania infantum parasites metastasize from the site of skin infection via unknown mechanisms, and establish infection within visceral organs usually without inducing skin pathology, resulting in the potentially fatal disseminated disease, Visceral Leishmaniasis (VL). Mouse studies suggest early responses at the skin infection site are critical determinants of subsequent adaptive immune responses in leishmaniasis, yet few studies address the role of keratinocytes, the most abundant immunoactive cell in the epidermis. We hypothesize that Leishmania infection causes keratinocytes to produce immunomodulatory factors that influence the outcome of infection.
Incubation of primary or immortalized human keratinocytes with L. infantum or L. major elicited dramatically different responses. Keratinocytes incubated with L. infantum significantly increased expression of pro-inflammatory genes IL6, IL8, TNF, and IL1B by RT-qPCR; whereas keratinocytes exposed to L. major did not. Similar to live parasites, L. infantum-derived exosomes induced more IL8 mRNA compared to control or L. major-derived exosomes. Western blotting confirmed NFkBp65 phosphorylation in keratinocytes exposed to L. infantum but not L. major. However, no evidence of L. major inhibition of TNF-induced NFkBp65 phosphorylation was observed in simultaneously treated keratinocytes. To examine whether keratinocytes influence proximal host cells, L. infantum-infected human monocytes were co-cultured with keratinocytes across a transwell membrane. These studies suggested L. infantum-exposed keratinocytes release soluble factors that enhance monocyte control of intracellular L. infantum replication. L. major-exposed keratinocytes had no comparable effect. These data suggest L. infantum and L. major differentially activate keratinocytes to release factors that limit infection in monocytes.
Microarray analyses performed on human keratinocytes exposed to either L. infantum or L. major promastigotes identified a limited number of transcripts increased by parasite exposure. Consistent with RT-qPCR observations, several inflammatory cytokine and chemokine genes were more strongly induced in L. infantum-exposed keratinocytes compared to L. major-exposed keratinocytes. Pathway analyses of genes induced by L. infantum-treated keratinocytes suggested that this interaction may induce neutrophil recruitment. Notably, AP1 transcription factor subunit genes were significantly down regulated in L. major-treated compared with L. infantum-treated or control keratinocytes. This suggests L. major may actively inhibits this keratinocyte activation, which might affect its ability to establish infection within host skin. In addition, ex vivo intradermal infection of human skin explants was explored as a method to compare keratinocyte responses to L. infantum or L. major in the context of whole skin tissue and the effects of vector salivary gland components are considered.
The response of keratinocytes found in these studies using L. infantum and L. major may give insight into the local host pathologic responses to different Leishmania species leading to visceralizing versus cutaneous manifestations to infection. We propose that Leishmania spp. elicit or evade a pro-inflammatory response by keratinocytes at the site of infection, generating a microenvironment uniquely tailored to each Leishmania species.
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Adamson, Rachel Elizabeth. "Molecular studies of Venezuelan sandflies and Leishmania isolates". Thesis, University of Liverpool, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385097.
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Lara, Diana. "Cationic steroid antibiotics as potential chemotherapeutic agent against Trypanosoma cruzi and Leishmania major". To access this resource online via ProQuest Dissertations and Theses @ UTEP, 2007. http://0-proquest.umi.com.lib.utep.edu/login?COPT=REJTPTU0YmImSU5UPTAmVkVSPTI=&clientId=2515.
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Thalhofer, Colin Joseph. "Leishmania infantum chagasi induces a dynamic cellular inflammatory response". Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/1091.
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Leishmania infantum chagasi (Lic) is a pathogenic protozoan parasite and one of the etiological agents of human visceral leishmaniasis (VL). VL is a potentially deadly disease characterized by variable fevers, cachexia, hepatosplenomegaly, and global immune suppression. Many questions regarding the pathogenesis of VL and the mechanisms of host defense during Lic infection remain to be elucidated. The primary focus of this thesis is the relationship between Lic and the mammalian immune system. We studied parasite-host interactions during Lic infection at the molecular, cellular, and organismal level. We generated transgenic parasites that expressed firefly luciferase and/or fluorescent proteins to expand our capacity to detect, observe, and quantify the parasites in a variety of experimental settings with modern analytical methodologies. Using luciferase-expressing Leishmania, we developed an experimental infection model in which parasites were detected and the relative parasite burden in specific anatomical locations could be quantified in a live animal host using bioluminescence imaging. This method allowed the parasite burden to be assessed in the same host throughout the course of infection. Utilizing this model we have made some intriguing observations relating to the kinetics and distribution of the parasite burden over time. The parasite burden was observed primarily in the liver and bone marrow over the first few weeks and then shifts to the spleen and bone marrow. To gain a better understanding of the initial parasite-host immune interactions in vivo, we studied the early inflammatory response after intradermal (i.d.) inoculation. We observed a rapid and abundant influx of neutrophils into the inoculated ears. The neutrophil influx was transient, dose dependent and specific for the local inoculation site. While there was not a significant neutrophil influx into the draining lymph nodes (dLN), there was an increase in the total cellularity and a striking increase in the relative proportion of B cells to T cells over the first week after intradermal parasite challenge. By inoculating transgenic mCherry-Lic we found that neutrophils were the primary parasite-laden host cell in the dermal tissue during the first day, but macrophages harbored most of the parasites by 2 days. Neutrophil depletion using low-dose antibody treatment resulted in a reduced rate of parasite uptake initially at the site of inoculation, but no significant change in the dLN dynamics. We further examined the parasite-host relationship by studying molecular signaling and cellular interactions between Leishmania and human neutrophils. We investigated the nature of the chemotactic activity of Leishmania-conditioned growth medium for human neutrophils by testing physical properties of the activity and ruled out some of the major Leishmania surface molecules as potential candidates. We aim to identify the agent(s) responsible for the activity in on-going studies. To this end, we are collaborating with a group at the NIH and testing biochemical purification/separation samples. We conclude that intradermal Lic challenge induces a rapid innate immune response at the local site of infection, that neutrophils sense Leishmania-derived factors leading to directed migration, and that neutrophils function as a primary site for Leishmania entry into the mammalian host.
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Solano, Gallego Laia. "Leishmania infantum and dog: immunological and epidemiological studies about infection and disease". Doctoral thesis, Universitat Autònoma de Barcelona, 2001. http://hdl.handle.net/10803/5719.
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L'objectiu d'aquesta tesi va ser obtenir nous coneixements sobre la immunologia i epidemiologia de la infecció per Leishmania en gossos que viuen en zones endèmiques. El capítol 2 descriu l'expressió d'anticossos anti-Leishmania IgG, IgG1 i IgG2 en una població de gossos. Els nivells d'immunoglobulines en gossos asimptomàtics van ser molt variables i menors que els dels animals malalts. En els animals malalts, els nivells de IgG i IgG2 van ser molt alts però els nivells d' IgG1 molt variables. Els resultats van mostrar la gran variació en l'expressió de IgG1 tant en gossos asimptomàtics com en simptomàtics, així com la baixa correlació de la IgG1 amb la IgG o IgG2.
El capítol 3 descriu l'avaluació i comparació de l'eficàcia de dues preparacions de leishmanines en la detecció de la resposta cel·lular davant de Leishmania en el gos.
El capítol 4 mostra l'estudi de la resposta humoral i cel·lular en una població de gossos. El 77% dels gossos va mostrar una resposta immunitària específica davant de Leishmania, ja fos humoral o cel·lular. El 80% dels cans eivissencs i el 48% dels gossos d'altres races van presentar resposta cel·lular. Els resultats van mostrar que la taxa d'infecció era molt alta, i que els gossos presentaven un ampli ventall de respostes inmunitàries, des de gossos resistents fins a gossos malalts. El ca eivissenc va manifestar més uniformement resposta cel·lular.
El capítol 5 descriu l'estudi de la prevalença de la malaltia, la seroprevalença i la prevalença de la infecció. Es va analitzar el quadre clínic, la serologia i la presència d'ADN de Leishmania en diferents teixits. La prevalença de la malaltia i seroprevalença van ser, respectivament, d'un 13% i d'un 26%. Els resultats de PCR positives en moll d'ós, conjuntiva i pell van ser respectivament, d'un 18%, 32% i 51%. La prevalença de la infecció va ser d'un 67%. La majoria de gossos de zona endèmica ha estat infectat pel paràsit.
El capítol 6 descriu l'estudi de paràmetres immunològics (serologia, test intradèrmic amb leishmanina, assaig de proliferació de limfòcits i detecció d'IFN-g i TNF-a) en l'avaluació de gossos infectats amb Leishmania. La majoria dels animals infectats sense simptomatologia clínica presentava nivells variables d'anticossos, reacció positiva al test intradèrmic, bona resposta proliferativa al antigen de Leishmania i producció d'IFN-g. La resta va mostrar nivells variables d'anticossos però absència de reacció al test intradèrmic. Abans del tractament, els animals malalts presentaven alts nivells d'anticossos, test intradèrmic negatiu, no producció d'IFN-g i producció de TNF-a. La millora clínica es va associar amb la disminució dels anticossos i amb l'augment del diàmetre del test intradèrmic. La combinació de la serologia, el test intradèrmic i la medició de citocines constitueixen tècniques útils i d'alta rellevància clínica en l'avaluació de la resposta inmunitària d'un pacient individual.
The objective of this thesis was to obtain new knowledge about the immunology and epidemiology of Leishmania infection in dogs living in endemic regions in the Mediterranean basin.
Chapter 2 describes the expression of IgG, IgG1 and IgG2 specific antibodies to L. infantum in a wide population of dogs. The levels of immunoglobulins in asymptomatic dogs were highly variable and lower than those found in ill dogs. In ill dogs, the levels of IgG and IgG2 were very high but the levels of IgG1 were extremely variable. Overall results showed a large variation in the IgG1 expression in asymptomatic and symptomatic dogs and a low IgG1 correlation with IgG or IgG2.
Chapter 3 describes the evaluation and comparison of the efficacy of two leishmanins for detection dog Leishmania cellular immune response.
Chapter 4 describes the study of humoral and cellular responses in a population of dogs. Seventy-seven percent of the dogs demonstrated a specific Leishmania response either humoral or cellular. Eighty percent of ibizian hounds and 48% of dogs of other breeds presented a cellular response. The results showed that the rate of infection was high and that dogs presented a broad range of immune responses from resistant to ill dogs. The Ibizian hound manifested a more uniform cellular response.
Chapter 5 describes the study of the prevalence of Leishmania infection, the seroprevalence and the prevalence of canine leishmaniosis. Clinical exploration for the presence of clinical signs compatible with leishmaniosis, the titre of anti-Leishmania antibodies, and the presence of Leishmania DNA by PCR on several tissues were assessed. The prevalence of the disease was 13% and the seroprevalence was 26%. The results of positive PCR in the bone marrow, the conjunctiva and the skin were 18%, 32% and 51%, respectively. The prevalence of the infection was 67%. The results showed that Leishmania infects the majority of dogs living in an endemic area.
Chapter 6 describes the study of immunological parameters (anti-Leishmania IgG1, IgG2, total IgG antibodies, LST, LPA, and production of IFN-g and TNF-a) in the evaluation of dogs infected by Leishmania. The majority of infected animals without clinically patent disease showed variable titres of anti-Leishmania antibodies, a positive LST, a strong Leishmania antigenic proliferative response, and a high production of IFN-g. The remainder showed positive titres of anti-Leishmania antibodies with a negative positive LST. Before treatment, ill dogs presented high levels of anti-Leishmania antibodies, negative LST, no production of IFN-g but a production of TNF-a. Clinical recovery was associated with a decrease in the titre of antibodies and an increase of the diameter of the LST. The combination of serology, LST, and measurement of cytokines constitutes a useful, clinically relevant method to evaluate the immune response to Leishmania in a single patient.
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Depledge, Daniel Pearce. "Comparative genomic analysis of three Leishmania species that cause diverse disease pathologies". Thesis, University of York, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.495870.
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In 1940, Virginia Woolf called for a more inclusive form of biography, which would include ‘the failures as well as the successes, the humble as well as the illustrious’. She did so partly as a reaction against Victorian biography, deemed to have been overly preoccupied with the great and the heroic. Yet a significant number of Victorian biographers did in fact write biographies that went against the trend of hero-worshipping ‘Great Lives’ and focused instead on the humble, the marginal, or the neglected. Though many are simplistic, pious productions, others sought to engage in contemporary debates surrounding the role and place of the individual in society in a sophisticated and complex manner. The thesis begins with an overview of the period’s biographical writings. The second and third chapters explore the representation of marginality and powerlessness through biographies of female and working-class subjects. The fourth and fifth chapters are concerned with issues of canonisation: the championing of neglected artists, and the Dictionary o f National Biography are discussed. A final, brief, chapter on Virginia W oolfs conception of ‘obscure lives’ seeks to broaden our understanding of her literary influences. The ‘obscure’ biographical subject emerges as a paradoxical figure used as a safe means of exploring the boundary between the private and the public. Above all, and in contrast with the trend instigated by Woolf, biographers were not concerned with securing immortality for their subjects, but with prompting within their readers feelings of empathy and gratitude. The thesis attempts to balance a survey of this trend with close analysis of works that manipulated the biographical genre in interesting ways. It is also a study of how Thomas Carlyle’s and George Eliot’s influence was disseminated within an under-studied literary genre. The thesis includes, as an appendix, a descriptive catalogue of over two hundred Victorian biographies.
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Tezuka, Daiane Yukie. "Triagem de compostos anti-chagásicos com o Trypanosoma cruzi e leishmanicidas com as espécies Leishmania amazonensis e Leishmania chagasi". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-25112015-101003/.
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Infecções causadas por tripanossomatídeos causam milhares de mortes anualmente, além de levarem a redução da capacidade produtiva, com elevada morbidade na população acometida. A eficácia terapêutica é limitada na maioria dos casos, sendo o benzonidazol o único fármaco aprovado para uso do tratamento do Trypanosoma cruzi, sendo ativo somente na fase aguda da Doença de Chagas, com grande efeitos colaterais. No caso das leishmanioses, as substâncias de tratamento existentes causam toxicidade renal e cardíaca, além de induzirem a resistência e apresentarem eficácia insuficiente. Observando todo o contexto torna-se necessário a busca por novas substâncias que sejam mais eficazes e menos tóxicas. Assim, o trabalho representa uma contribuição para a busca de novas moléculas bioativas para o tratamento da doença de Chagas e leishmaniose a partir da padronização e realização de ensaios celulares usando a forma epimastigota da cepa Y do T. cruzi e promastigota para Leishmania chagasi e L. amazonensis. A padronização do método colorimétrico de MTT foi realizada a partir da comparação com o método tradicional de contagem por microscopia usando câmara de Neubauer, seguido do estudo de viabilidade por citometria de fluxo, como método confirmatório e, finalmente a padronização do ensaio de ciclo celular usando os fármacos de referência benzonidazol (T. cruzi) e anfotericina B (Leishmania spp.). Os compostos testados neste estudo são proveniente de classes distintas de inibidores de alvos macromoleculares (cisteíno proteases, DHODH, GAPDH, quinases) e foram planejados e/ou selecionados pelo Grupo de Química Medicinal (NEQUIMED). Dentre todas as substâncias, inibidores de quinases apresentaram maior potencial para estudos subsequentes, sendo o T. cruzi o parasito mais sensível, onde um grande número de substâncias apresentou atividade nos estudos de triagem. Para uma delas (Neq0474) foi realizado o ensaio de dose-resposta, com EC50 = 53 µmol/L. L. chagasi apresentou a maior resistência dentre todos os parasitos estudados neste trabalho, enquanto que L. amazonensis foi sensível para Neq0438. Algumas substâncias estudadas apresentaram potencial para estudos mais aprofundados visando identificar novas alternativas terapêuticas para estas doenças parasitárias.Infections caused by trypanosomatides lead to thousands of deaths annually, besides the reduction of the quality of life and working capability, with high morbidity to the patients. The therapy efficacy is limited in most cases, being benznidazole the only approved drug in Brazil, which works only in the acute phase of the Chagas Disease with severe collateral effects. For leishmaniasis, the drugs cause renal and cardiac toxicity and trigger resistance. In this context, novel efficacious and secure substantes are necessary to improve the current therapeutic strategies, which was the goal of this project by means of cell-based assays. The first step was the standardization of the MTT colorimetric assay for the epimastigote form of the Y strain of Trypanosoma cruzi and promastigote form of Leishmania chagasi and Leishmania amazonensis. This was achieved by comparing the results with the standard counting using the Neubauer chamber. The use flow cytometry for the determination of cell viability and the perturbation of the cell cycle were also standardized using the reference drugs benznidazole (T. cruzi) and amphotericin B (Leishmania spp.). New compounds tested in this project were designed or selected based on different macromolecular targets (cysteine proteases, DHODH, GAPDH, kinases) by the Medicinal Chemistry Group (NEQUIMED). Among them, many kinase inhibitors promoted the most dramatic results for T. cruzi, reducing the cell viability of this parasite. One of them (Neq0474) was subjected for a follow-up dose-response assay, with EC50 = 53 µmol/L. L. chagasi was the most resistant parasite in this work, whereas L. amazonensis was sensitive to Neq0438. Some of these new compounds are of interest for more in-depth studies to these parasitic diseases.
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Alessi, Claudia Alvares Calvo. "Leishmaniose cutânea americana no Pontal do Paranapanema - SP: avaliação clínica, histopatológica e uso da reação em cadeia da polimerase (PCR) para identificação e caracterização das espécies de Leishmania". Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/5/5144/tde-17022009-115336/.
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As leishmanioses são doenças parasitárias causadas por protozoários do gênero Leishmania e são importante problema de saúde pública. A leishmaniose cutânea americana é considerada doença autóctone do continente americano e se apresenta com diversas formas clínicas, que dependem da espécie que causa a infecção e de outros fatores como virulência e capacidade de evasão do sistema immune. São reconhecidas seis espécies de Leishmania que causam casos humanos de LCA no Brasil, destas, cinco pertencem ao subgênero Viannia e uma ao subgênero Leishmania. Elas são: Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) shawi, Leishmania (Viannia) naiffi e a Leishmania (Leishmania) amazonensis. A transmissão da leishmaniose cutânea se mantém na região do Pontal da Paranapanema, com 20 casos notificados em 2006. A Leishmania (V.) braziliensis é a única espécie considerada como agente da doença na região, com identificação dos vetores envolvidos e de possíveis reservatórios silvestres. O objetivo do trabalho é o estudo dos aspectos epidemiológicos, clínicos e histopatológicos da leishmaniose cutânea no Pontal do Paranapanema e a identificação, por métodos moleculares, PCR, do agente etiológico e a caracterização do gênero, subgênero e a espécie de Leishmania presentes na região. A doença foi encontrada em ambos os sexos, predominando no sexo masculino (67,9%), em todas as faixas etárias, mas 70,5% estavam na faixa de 20 a 49 anos de idade. A forma clínica mais encontrada foi a cutânea, com 92,3% dos casos. A pesquisa de parasita na lesão em 78 pacientes que realizaram biópsias foi positiva em 40 amostras (51.3%), em lâminas coradas pela HE; quando se utilizou a IH foi 66,7%. O índice de concordância entre as técnicas da HE e IH foi de 58,97%. Entretanto, 10 casos negativos na IH foram positivos na HE, e de 38 casos negativos na HE, 22 foram positivos na IH. Isto mostra que há necessidade de associação dos dois métodos. A positividade na PCR foi de 53,8%. Avaliando-se os resultados obtidos nesse estudo, podemos verificar que dos 40 casos positivos pela HE, 24 também foram positivos pela PCR; porém, 16 destes, foram negativos pela PCR. Em contrapartida, das 38 amostras negativas na HE, 18 delas foram positivas pela PCR. Pela imunohistoquímica, do total de 26 amostras negativas, apenas 12 permaneceram negativas e 14 foram positivas na PCR; enquanto que, das 52 amostras positivas pela IH, 28 foram positivas e 24 negativas pela PCR. Os níveis de concordância da PCR com HE foram de 56,41% e da PCR com IH de 51,28%. Esses resultados reforçam a idéia da necessidade de se associar os três métodos para o diagnóstico da LC. As características das lesões histopatológicas foram: reação granulomatosa (RG) encontradas em 71,85%, reação granulomatosa com células gigantes (RGCG) em 12,8%, reação granulomatosa com necrose (RGN) em 10,3% e reação granulomatosa com necrose e células gigantes (RGNCG) em 5,1% dos casos. Utilizando-se os primers SSU rDNA S17/S18, foi possível caracterizar, através do seqüenciamento, 27 (34,6%) amostras como sendo do subgênero Viannia e 06 amostras como L. (L.) amazonensis. Este estudo identificou o primeiro caso de L. (L.) amazonensis na regiãoLeishmaniasis are parasitic diseases caused by protozoans of the Leishmania genus and are important public health problems. American cutaneous leishmaniasis (ACL) is considered an autochthonous disease of the American continent and presents several clinical forms which depend on the causative species of the infection and other factors such as virulence and ability to evade the immune system. Six Leishmania species are recognized to cause human ACL cases in Brazil of which five belong to the Viannia and one to the Leishmania subspecies. They are: Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) shawi, Leishmania (Viannia) naiffi and Leishmania (Leishmania) amazonensis. Cutaneous leishmaniasis transmission is maintained in the Pontal do Paranapanema region, with 20 notified cases in 2006. Leishmania (V.) braziliensis is the only species considered to be the disease agent in the region with identification of the involved vectors and possible wild reservoirs. The aim of this research is the studies of the epidemiological, clinical and histopathological aspects of cutaneous leishmaniasis in the Pontal do Paranapanema and the identification by molecular methods, PCR, of the etiologic agent and characterization of the Leishmania genus, subgenus and species present in the region. The disease was found in both genders, with predominance of males (67.9%), in all age ranges, but 70.5% were in the range of 20 to 49 years. The cutaneous was the mostly found clinical form with 92.3% of the cases. Search for the parasite in the lesion of 78 patients who underwent biopsies was positive in 40 samples (51.5%), in HE stained slides; when IH was used, 66.7% were positive. Agreement index between the HE and IH techniques were 58.97%. However, 10 negative cases using IH were positive with HE, and of 38 HE negative cases 22 were positive using IH. This shows that association of the two methods is needed. Using PCR, there was a positivity of 53.8%. Evaluating the results obtained in this study, we may observe that of the 40 HE positive cases 24 were also positive on PCR; but 16 of these were PCR negative. Contrariwise, of the 38 HE negative samples 18 were positive PCR. Using immunohistochemistry, of the total of 38 HE negative samples, 18 were positive with PCR; while of the 52 IH positive samples, 28 were positive and 24 negative on PCR. Agreement levels of PCR with HE were 56.41%, and of PCR with IH 51.28%. These results reinforce the idea of the need for association of the three methods for CL diagnosis. Histopathological lesion characteristics were: granulomatous reaction (GR) found in 71.85%, granulomatous reaction with giant cells (GRGC) in 12.8T, granulomatous reaction with necrosis (GRN) in 10.3% and granuloma with necrosis and giant cells (GRNGC) in 5.1% of the cases. Using SSU rDNAS 17/S18 primers it was possible to characterize through sequencing 27 (34.6%) samples as being of the Viannia subgenus and 06 samples of the L. (L.).amazonensis This study identified the first L. (L.) amazonensis case in the region
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Karam, Marc Christophe. "The role of cytokines in the regulation of hyperalgesia and disease progression in Leishmania major infection". Thesis, University of Surrey, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.431123.
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Pereira, Lucas Campana. "Busca de genes associados à resposta ao teste de Montenegro para antígenos de Leishmania". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-27022013-155152/.
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O presente trabalho visa, através de métodos genético-epidemiológicos, identificarem genes associados à resposta ao antígeno da Leishmania. Utilizando amostras através do teste de Montenegro dos municípios de Monte Negro (RO) e Assis Brasil (AC). Na primeira abordagem foram feitos testes com TaqManÒ e a segunda com GWAS, e análises de associação foram feitas utilizando-se o pacote SPSS e o Plink. Não foram encontradas associações com cinco SNPs (MYD88, IL12, IL10, IFNGR1 e NRAMP1). A análise de dados de varredura genômica com filtros, indicou uma região no cromossomo 10 com 3 SNPs próximos que fazem parte de uma região regulatória que com o posterior auxilio do real time não se confirmaram, apesar do ensaio RS11251056 apresentar valores limítrofes, se tornando uma possível indicação para trabalhos futuros e por fim a último teste foi a meta-análise, através do método de Woolf, apresentou resultados indicativos de associação para ensaios encontrados no cromossomo 2 com ZNF638 relacionados a diferenciação celular e também no cromossomo 10 que contem lincRNAs e o gene NGR3, com dois ensaios apresentando valores significativos de p, onde podemos inferir que estas duas regiões podem participar ativamente na diferenciação da resposta ao antígeno da Leishmania.The present study aims, through genetic-epidemiological methods, to identify genes associated with response to Leishmania antigen. Using samples Montenegro skin test through the municipalities of Monte Negro (RO) and Assis Brazil (AC). In the first approach were tested with TaqManÒ and the second GWAS, and association analyzes were performed using SPSS and Plink. No associations were found with five SNPs (MyD88, IL12, IL10, IFNGR1, and NRAMP1). The analysis of genome scan data with filters, indicated a region on chromosome 10 with three nearby SNPs that are part of a regulatory region that later with the help of real time is not confirmed, although the test rs11251056 have borderline values, becoming an possible direction for future work and finally the last test was the meta-analysis by the method of Woolf, presented results indicating the association found in tests for chromosome 2 with ZNF638 related to cell differentiation and also on chromosome 10 that contains lincRNAs and gene NGR3, two runs with a significant p value, where we can infer that these two regions can actively participate in the differentiation of the response to Leishmania antigen.
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Yakovich, Adam J. "Old targets and new beginnings a multifaceted approach to combating Leishmaniasis, a neglected tropical disease /". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1193247442.
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Santos, Marcos André Ferreira. "Cytokine gene expression and cellular immune response in dogs with leishmaniosis before and under the two first-line treatment protocols : new insights into the animal disease". Doctoral thesis, Universidade de Lisboa, Faculdade de Medicina Veterinária, 2020. http://hdl.handle.net/10400.5/20540.
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Tese de Doutoramento em Ciências Veterinárias, especialidade de Ciências Biológicas e Biomédicas. Universidade de Lisboa. Faculdade de Medicina Veterinária 2020Canine leishmaniosis (CanL) caused by Leishmania infantum is a zoonotic visceral disease of worldwide concern. The drugs used for its treatment improve the animal’s clinical condition, although, in most cases, the parasites are not completely destroyed. The current study aimed to evaluate the immune response of the dog with leishmaniosis before and during treatment with first-line drugs, by analyzing the profile of cytokines and subsets of CD4+ and CD8+ T-cells in peripheral blood, lymph node and bone marrow. Two groups of six dogs diagnosed with CanL were treated with either miltefosine or meglumine antimoniate in combination with allopurinol. Simultaneously, another group of ten clinically healthy dogs was used as a control group. Upon diagnosis and during the following three months of treatment, clinical signs, hematological and biochemical parameters, urinalysis results and anti-Leishmania antibody titers using IFAT were recorded. Furthermore, peripheral blood, popliteal lymph node and bone marrow mononuclear cells were collected to evaluate the gene expression of IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β and IFN-γ by qPCR. In parallel, these cells were also immunophenotypically analyzed be flow cytometry, using surface monoclonal antibodies anti-CD45, CD3, CD4, CD8, CD25 and intracellular monoclonal antibody anti-nuclear factor FoxP3. Both treatment protocols promoted the remission of clinical signs, normalization of hematological and biochemical parameters and urinalysis values. Sick dogs showed a generalized increase in IFN-γ gene expression and a decrease of IL-2, IL-4, and TGF-β. The expression of IL-12, TNF-α, IL-5, and IL-10 showed variations between groups of dogs and the tissue analyzed. CanL also resulted in an overall increase in the percentage of CD8+ T-cells in all tissues. In the peripheral blood there was also a decrease in CD4+ T-cells and an increase of CD4+CD25+FoxP3+ and CD8+CD25+FoxP3+ T-cells, with the latter also increasing on the bone marrow. CD4+CD25-FoxP3- T-cells showed a marked decrease in blood and bone marrow. During treatment, a trend towards normalization of cytokine gene expression and T-cell subsets was observed. However, high levels of IFN-γ gene expression were still observed in all tissues. In turn, the treatments caused an increase in the percentage of CD4+CD25+FoxP3+ and a decrease in CD8+CD25-FoxP3- T-cells, leading to normalization of CD4+ and CD8+ T cells in all tissues. Furthermore, the effect of treatment on gene expression of cytokines that were not significantly altered by infection indicates that these combined treatment protocols directly affect cytokine production. Both combined treatments are effective in remitting clinical sings and appear to influence the dog’s immune response, sustaining a pro-inflammatory immune environment while promoting the normalization of T-cell subsets. These findings indicate that L. infantum may be able to manipulate elements of the dog's immune system to avoid differentiating an efficient protective response, preventing the rapid development of severe pathology while ensuring the parasite’s survival and securing the possibility of several transmission cycles. Allied to these results, other studies carried out in collaboration with the working group on the role of neutrophils, hepatocytes and Kupffer cells in CanL, as well as the evaluation of treatment in feline leishmaniosis, have allowed to enhance the knowledge in the area of animal leishmaniosis.
RESUMO - Expressão génica de citocinas e resposta imune celular em cães com leishmaniose antes e sob os dois protocolos de tratamento de primeira linha: novas informações sobre a doença animal - A leishmaniose canina (LCan) causada por Leishmania infantum é uma doença visceral zoonótica de interesse mundial. Os fármacos utilizados para o tratamento melhoram o estado clínico do animal, embora, muitas das vezes, os parasitas não sejam totalmente eliminados. O presente trabalho teve como objetivo avaliar a resposta imunitária do cão com leishmaniose antes e durante o tratamento com fármacos de primeira linha, através da análise do perfil de citocinas e subconjuntos de células T CD4+ e CD8+ no sangue periférico, linfonodo e medula óssea. Dois grupos de seis cães diagnosticados com LCan foram tratados com antimoniato de meglumina ou miltefosina em associação com alopurinol. Em simultâneo, outro grupo de dez cães clinicamente saudáveis foi usado como grupo controlo. Aquando do diagnóstico e durante os três meses consecutivos de tratamento, foram registados os sinais clínicos, parâmetros hematológicos e bioquímicos, resultados de urianálise e títulos de anticorpos anti-Leishmania obtidos por IFAT. Células mononucleares do sangue periférico, linfonodo e medula óssea foram recolhidas para avaliação da expressão génica de IL-2, IL-4, IL-5, IL-10, IL-12, TNF-α, TGF-β e IFN-γ por qPCR. Em paralelo, estas células foram também analisadas imunofenotipicamente por citometria de fluxo, com anticorpos monoclonais de superfície anti-CD45, CD3, CD4, CD8, CD25 e anticorpo mononuclear intracelular anti-factor nuclear FoxP3. Ambos os protocolos de tratamento promoveram a remissão dos sinais clínicos, a normalização dos parâmetros hematológicos, bioquímicos e dos valores de urianálise. Cães doentes mostraram um aumento generalizado da expressão génica de IFN-γ e diminuição de IL-2, IL-4 e TGF-β. A expressão de IL-12, TNF-α, IL-5 e IL-10 apresentou variações entre os grupos de cães e o tecido analisado. A LCan levou também a um aumento generalizado da percentagem de células T CD8+ em todos os tecidos. No sangue verificou-se ainda diminuição de células T CD4+ e aumento de células T CD4+CD25+FoxP3+ e CD8+CD25+FoxP3+, com estas últimas aumentando também na medula. As células CD4+CD25-FoxP3- mostraram diminuição acentuada no sangue e medula óssea. Durante o tratamento, foi observada uma tendência para a normalização da expressão génica de citocinas e subconjuntos celulares. No entanto, níveis elevados da expressão génica de IFN-γ foram observados em todos os tecidos. Por sua vez, os tratamentos causaram um aumento da percentagem de células T CD4+CD25+FoxP3+ e diminuição de células T CD8+CD25-FoxP3-, levando à normalização os valores de células T CD4+ e CD8+ em todos os tecidos. Adicionalmente, o efeito do tratamento na expressão génica de citocinas, que não se encontravam alteradas aquando da infeção, é indicador de que estas terapêuticas combinadas afetam diretamente a produção de citocinas. Ambas as terapêuticas combinadas são eficazes na remissão dos sinais clínicos e parecem influenciar a resposta imunitária do cão, sustentando um ambiente imunológico pró-inflamatório e promovendo a normalização de subconjuntos de linfócitos T. Estes resultados indicam que L. infantum poderá ser capaz de manipular elementos do sistema imunológico do cão para impedir a diferenciação de uma resposta protetora eficaz, evitando o rápido desenvolvimento de patologia grave, enquanto assegura a sobrevivência do parasita, garantindo a possibilidade de vários ciclos de transmissão. Aliado a estes resultados, estudos realizados em colaboração pelo grupo de trabalho sobre o papel dos neutrófilos, hepatócitos e células de Kupffer na LCan, assim como a avaliação do tratamento na leishmaniose felina, permitiram agregar mais conhecimentos na área da leishmaniose animal.
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Santos, Priscilla Vargas Walsh Gonçalves dos. "Relação imunogenética dos pênfigos com a leishmaniose tegumentar". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17138/tde-20072016-144443/.
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Pênfigo é uma dermatose bolhosa autoimune, endêmica em algumas áreas, como no nordeste do estado de São Paulo, Brasil, caracterizada pela produção de autoanticorpos contra desmogleínas (Dsg) - proteínas de adesão dos queratinócitos. O pênfigo vulgar (PV) acomete mucosas e pele, pela produção de anti-Dsg 3 e 1, respectivamente. O pênfigo foliáceo (PF) apresenta lesões exclusivamente na pele, pela produção de anti-Dgs1. Esta região também é endêmica para a Leishmaniose Tegumentar Americana (LTA), cujo principal fator etiológico é Leishmania (Viannia) braziliensis. Objetivos: Relacionar fatores imunogenéticos dos pênfigos com aqueles da LTA, investigando, em pacientes com pênfigos, LTA e em controles: a resposta humoral às Dsg 1 e 3 e contra Chagas; a resposta humoral e celular aos antígenos de leishmania; e a associação dos alelos HLA de classe II -DR e -DQ no grupo LTA com aqueles de suscetibilidade e de resistência descritos nos pênfigos. Métodos: A resposta humoral foi investigada por: (i) ELISA para determinação dos anticorpos anti-Dsg 1 e 3, anti-L. V. braziliensis e contra T. cruzi; (ii) imunoblotting (IB) com extrato proteico de epiderme e com extrato proteico de L. (V.) braziliensis; (iii) imunofluorescência indireta (IFI) com substrato de pele humana e soro de pacientes com LTA. A resposta celular foi realizada por teste intradérmico de Montenegro (TIM). A tipificação dos alelos HLA -DR e -DQ foi realizada por PCR-SSOP. Resultados: A resposta humoral às Dsg confirmou o esperado - anti-Dsg1: 84,6% no grupo PF e 54,8% no grupo PV; e antiDsg3: 83,9% no PV; não havendo diferença significativa entre os grupos LTA e controles - anti-Dsg1: 16% nos familiares de PF (FPF); 5,2% no grupo LTA; 4,2% no grupo FPV; e 2,7% nos controles vizinhos; e anti-Dsg3: 12% no grupo FPF; 6,4% no grupo PF e 5,2% no grupo LTA. Houve reconhecimento dos peptídeos de 130kDa (corresponde ao PM da Dsg3), 145kDa, 150kDa (Dsg2), 160kDa (Dsg1), 230kDa (BP230), 250kDa, 290kDa, 350kDa, 410kDa, 425kDa e 460kDa da epiderme humana por soro de pacientes com LTA. A IFI resultou positiva para anti-IgG em 2/6 pacientes com LTA. Em um deles, houve reconhecimento de peptídeos intercelulares da epiderme, guardando semelhança aos pênfigos; e, no outro, de antígenos da zona da membrana basal, guardando semelhança ao penfigoide bolhoso. A resposta humoral a L. (V.) braziliensis resultou mais elevada no grupo LTA (73% para LTAc e 62% para LTAm) em relação aos demais grupos, sem diferença significativa entre os grupos pênfigos (1,3% no grupo PF e 1,6% no PV) e os controles (10,8% nos controles vizinhos e 4% no grupo FPF, FPV e controles Banco de Sangue). Pacientes com pênfigos apresentaram títulos sorológicos para T. cruzi semelhantes aos controles. Houve reconhecimento de peptídeos de L. (V.) braziliensis pelo soro dos pacientes com pênfigo (45kDa, 95kDa, 100kDa, 120kDa, 125kDa, 145kDa, 150kDa, 305kDa, 330kDa, 410kDa e >500kDa). O TIM foi negativo nos 6 pacientes com PF ou PV avaliados. Os alelos DRB1*01:02 e DQA1*01 mostraram associação de resistência para LTA e suscetibilidade para PF; o alelo DRB1*04:02 de resistência para LTA e suscetibilidade para PV; os alelos DRB1*07:01 e *11:01 de suscetibilidade para LTA e resistência para PF; e o DQA1*01:02 mostrou associação de suscetibilidade a ambos LTA e PF. Conclusões: os pacientes com LTA têm resposta humoral às Dsg 1 e 3, e os pacientes com pênfigo, aos antígenos de L. (V.) braziliensis e de T. cruzi semelhante aos controles. Os soros de pacientes com pênfigos reconhecem peptídeos da epiderme e da zona da membrana basal por IB e IFI. Os demais peptídeos reconhecidos pelos pacientes com LTA ao extrato epidérmico, assim como dos pacientes com pênfigos ao extrato de L. (V.) braziliensis necessitam sequenciamento. As associações de suscetibilidade ou resistência dos alelos HLA de classe II -DR e -DQ são opostas para LTA e pênfigo. Os resultados confirmam a não participação do parasito L. (V.) braziliensis na patogênese dos pênfigos, assim como corroboram a observação clínica da ausência da associação de ambas as doenças na região do estudoPemphigus is an autoimmune bullous dermatosis, endemic in some areas, such as in the northeastern of São Paulo state, characterized by the production of autoantibodies against desmoglein (Dsg) - keratinocytes adhesion proteins. Pemphigus vulgaris (PV) affects mucous membranes and skin, by the production of anti-Dsg 3 and 1, respectively. Pemphigus foliaceus (PF) affects only the skin, by the production of anti-Dsg 1. This region is also endemic for American Cutaneous Leishmaniasis (ACL), whose main etiological factor is Leishmania (V.) braziliensis. Objectives: To relate immunogenetic factors of pemphigus with those of ACL, investigating, in patients with pemphigus, ACL and controls: the humoral response to Dsg 1 and 3; the humoral and cellular responses to Leishmania antigens; and the association of class II -DR and -DQ HLA alleles in ACL group with those of susceptibility and resistance described in pemphigus. Methods: The humoral response was investigated by (i) ELISA for determination of anti-Dsg1 and anti-Dsg3 antibodies and anti-antibodies from T. cruzi and L. braziliensis; (ii) immunoblotting (IB) with protein extract of the epidermis and protein extract of L. braziliensis; (iii) indirect immunofluorescence (IIF) with human skin substrate and serum of ACL patients. The cellular response was carried out by intradermal test Montenegro (ITM). The typing of HLA-DQ and -DR alleles was performed by PCR-SSOP. Results: The humoral response to Dsg confirmed expected in groups of PV and PF (anti-Dsg1: 84.6% in the PF group and 54.8% in the PV group, and anti-Dsg3: 83.9% in PV), with no significant difference between the ACL and control groups [anti-Dsg1: 16% in relatives of PF (RPF), 5.2% in the ACL, 4.2% in relatives of PV (RPV) and 2.7% of controls neighbors; anti-Dsg3: 12% in RPF, 6.4% in the PF group and 5.2% in ACL)]. There has been recognition of peptides 130kDa, 145 kDa, 150kDa, 160kDa, 230 kDa, 250 kDa, 290 kDa, 350 kDa, 410 kDa, 425 kDa and 460 kDa of human epidermis by serum of ACL patients. The IFI was positive in 1/6 ACL patients evaluated. The humoral response to L. braziliensis resulted higher in ACL group (73% to ACLc and 62% to ACLm) compared to the other groups, with no significant difference between pemphigus groups (1.3% in the PF group and 1.6% the group PV) and controls (10.8% in neighboring controls and 4% in FPF, in FPV and BS controls). Patients with pemphigus have serological titers to T. cruzi similar to controls. There was L. braziliensis recognition of peptides by the patients with pemphigus (45 kDa, 95 kDa, 100 kDa, 120 kDa, 125 kDa, 145 kDa, 150 kDa, 305 kDa, 330 kDa, 410 kDa and> 500 kDa). The ITM was negative in 06 patients with PF or PV evaluated. The DRB1*01:02 and DQA1*01 showed resistance association for LTA and susceptibility to PF; allele DRB1*04:02 resistance to ACL and susceptibility to PV; the DRB1*07: 01 and *11:01 susceptibility to ACL and resistance to PF; and DQA1*01:02 showed susceptibility association with both ACL and PF. Conclusions: Patients with ACL have humoral response to Dsg 1 and 3, and pemphigus patients to L. braziliensis and T. cruzi antigens similar to controls. The peptides recognized by patients with pemphigus to L. braziliensis extract require sequencing. The susceptibility or resistance associations of class II -DR and -DQ HLA alleles are opposed to ACL and pemphigus. The results confirm the non-participation of the parasite L. (V.) braziliensis in the pathogenesis of pemphigus, as well as support the clinical observation of the absence of both diseases association
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Escobar, Taiane Acunha. "Presença de Leishmania sp em equinos de zona urbana de Uruguaiana, Rio Grande do Sul". Universidade Federal do Pampa, 2015. http://dspace.unipampa.edu.br:8080/xmlui/handle/riu/506.
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A Leishmaniose é uma doença parasitária infeciosa, causada por protozoários do gênero Leishmania e transmitida pelo vetor flebótomo. Caracterizada como Doença Tropical Negligenciada, acomete diversas espécies de mamíferos, sendo o cão, atualmente, o principal reservatório em área urbanas. Os equinos também podem ser infectados, especialmente quando estão em contato com reservatórios ou vetores. No município de Uruguaiana – RS há um expressivo número de equinos utilizados na tração de cargas e como meio de transporte, com constante movimentação dentro do perímetro urbano. Esses animais vivem em condições precárias, submetidos a trabalhos excessivos e má nutrição. Frente a estes fatores somados à atual situação epidemiológica da leishmaniose visceral canina no município, o presente estudo foi realizado com o objetivo de identificar a presença de Leishmania sp em equinos urbanos do município de Uruguaiana-RS. Para a condução do experimento foram utilizadas amostras sanguíneas de 192 equinos testadas em três técnicas: sorológica (ELISA), imunocromatográfica (TR-DPP) e molecular (PCR). Na técnica de ELISA foi utilizado soro, testado com o Kit Ensaio Imunoenzimático para Diagnóstico da Leishmaniose Visceral Canina – Bio-Manguinhos e para o teste imunocromatográfico Teste Rápido Dual Path Platform utilizaram-se amostras de sangue total. As reações de PCR, após extração de DNA de sangue periférico dos animais, foram realizadas com quatro pares de iniciadores distintos. Todas as amostras testadas apresentaram-se não reagentes nos ensaios imunológicos. Entretanto, com o emprego da técnica PCR, mais sensível, houveram amostras positivas. Dos quatro pares de iniciadores testados, 75 amostras foram positivas, 52 com pelo menos um dos pares. Contudo, ao analisarmos individualmente os iniciadores, 58,6% foram positivos para LITSV/L5.8SR, 44% para LITSV/LISTSR, 28% para RV1/RV2, e 4% LITSR/L5.8S. Os resultados apresentados no experimento indicam a possibilidade de existência de Leishmania em equinos na região de Uruguaiana, embora os testes sorológicos não tenham apresentado reatividade para Leishmania. A técnica molecular possibilitou a detecção do gênero Leishmania nas amostras de sangue periférico dos equinos. Este foi o primeiro relato da infecção na espécie equina na região do extremo oeste do estado do Rio Grande do Sul. Contudo, se faz importante a realização de sequenciamento do fragmento para que se possa confirmar a identidade genética de Leishmania sp.
Leishmaniasis is an parasitic infectious disease caused by protozoa genus Leishmania and transmitted by the sandfly vector. Characterized as Neglected Tropical Disease, affects several species of mammals, and the dog are main reservoir in urban areas. The horses can also be infected especially when they are in contact with reservoirs or vectors. In Uruguaiana’s city, there is a significant number of horses used in the tensile loads and means of transport, with constant movement within the city, living in precarious work’s conditions, subjected to excessive and poor nutrition. In view of these factors added to the current epidemiological situation of LVC in the city, the present study aim to identify the presence of Leishmania in urban horses the municipality of Uruguaiana-RS. For the experiment, blood samples from 192 horses were used for holding three techniques: serological (ELISA), immunochromatographic (TR-DPP) and molecular (PCR). For conducting ELISA’s Test serum was used and tested with Bio-Manguinhos kit and for TR-Dual Path Platform Rapid Test whole blood samples were employed. In the PCR technique, DNA was extracted from peripheral blood of animals and amplifications were performed with primers RV1 / RV2, LITSR / LITSV, LITSR / L5.8S / LITSV / L5.8SR.. All tested samples showed negative results in immunoassays. However, employing sensitive techniques such as PCR, positive samples were detected. Considering the four primer pairs tested, 75 animals were positive, 52 with at least one of the pairs. However, when analyzing the individual primers, 58.6% were positive for LITSV/L5.8SR, 44% to LITSV/LISTSR, 28% for RV1/RV2, and 4% LITSR/L5.8S.In The results presented in the experiment indicate the possibility of Leishmania in horses in the region of Uruguaiana city, although serological tests have not submitted reactivity to Leishmania. Molecular technich shows results to consider the Leishmania’s presence in horse’s peripheral blood samples. This was the first report of infection in equine species in westernmost region of the Rio Grande do Sul state. However, it is important to conduct sequencing of the fragment so that we can confirm the genetic identity of Leishmania sp.
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Davis, Richard Elliot. "Neutrophil responses to infection with leishmania parasites: MHC class II-expression and parasite life-stage interactions". Diss., University of Iowa, 2016. https://ir.uiowa.edu/etd/2200.
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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.
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Pinto, Erika Gracielle. "Isolamento, caracterização e atividade anti-leishmania chagasi e anti-trypanosoma cruzi de compostos bioativos de venenos de anfíbios brasileiros". Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/99/99131/tde-31012014-154317/.
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Dentre as doenças parasitárias tropicais, as causadas por protozoários se apresentam como um grande desafio para a saúde pública, sendo representadas pela leishmaniose e doença de Chagas. Afetam grandes populações marginais ao processo econômico globalizado, e desta forma, não são vistas como mercados potenciais. O presente projeto visou o isolamento de novos compostos naturais de venenos animais com atividade anti-Leishmania e anti-T. cruzi. O presente estudo fracionou por diferentes técnicas cromatográficas, os venenos dos anfíbios Siphonops annulatus, Corythomantis greeningi, Aparasphenodon brunoi e Phyllomedusa hypochondrialis, visando o isolamento de peptídeos e metabólitos secundários através de ensaios biomonitorados. Utilizando-se a espectrometria de massas e seqüenciamento por degradação química de Edman, foi possível a caracterização bioquímica de cinco peptídeos ativos da secreção de Phyllomedusa hypochondrialis, sendo estes a bradicinina, as dermaseptinas 1 e 4 e as filoseptinas 7 e 8. Os peptídeos apresentaram uma Concentração Efetiva 50% (CE50) variando entre 0,7 a 20 ?g/mL em L. (L.) infantum chagasi e T. cruzi, com baixa ou nenhuma citotoxicidade para células de mamíferos nas concentrações testadas. Além disso, a separação química da secreção do anfíbio Siphonops annulatus forneceu uma fração altamente ativa em promastigotas de L. (L.) infantum chagasi, com CE50 0,065 ?g/mL, porém com toxicidade bastante elevada para macrófagos peritoneais e nenhuma seletividade nas formas intracelulares. Estudos ultraestruturais de Leishmania demonstraram severos danos mitocondriais, além da formação de grande vacúolos citoplasmáticos, levando o parasita a morte em poucas horas. O presente estudo demonstrou o potencial de peptídeos e metabólitos secundários de venenos de anfíbios, que se adequadamente estudados, poderão contribuir como novos protótipos de fármacos para a doenças negligenciadas.Among the tropical parasitic diseases, those caused by protozoa present a major challenge to public health, being represented by leishmaniasis and Chagas disease. Affect large populations marginal to the global economic process, and thus are not seen as potential markets. This project aimed the isolation of new natural compounds in animal venoms with anti-Leishmania activity and anti-T. cruzi. The present study fractionated by different chromatographic techniques, the poisons of the amphibians Siphonops annulatus, Corythomantis greeningi, Aparasphenodon brunoi and Phyllomedusa hypochondrialis, aiming the isolation of peptides and secondary metabolites through bioguided assays. By using mass spectrometry and sequencing by Edman degradation, it was possible to do the biochemical characterization of five active peptides from the poison of Phyllomedusa hypochondrialis, as bradykinin, dermaseptins 1 and 4 and phylloseptins 7 and 8. The peptides showed a 50% Effective Concentration (EC50) ranging from 0.7 to 20 ?g/mL in L. (L.) infantum chagasi and T. cruzi, with little or no cytotoxicity to mammalian cells at the tested concentrations. In addition, the chemical separation of the poison of the amphibian Siphonops annulatus provided a highly active fraction against promastigotes of L. (L.) infantum chagasi, with an EC50 of 0.065 ?g/mL, but highly toxicity to peritoneal macrophages and without selectivity against the intracellular forms of Leishmania. Ultrastructural studies of Leishmania showed severe mitochondrial damages and the formation of large cytoplasmic vacuoles, leading to parasite death within few hours. The present study demonstrated the potential of peptides and secondary metabolites of amphibian poisons, and if adequately studied, may contribute as prototypes of new drugs for neglected diseases.
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Abbehusen, Melissa Moura Costa. "Imunização de cães com produtos oriundos de Lutzomyia Longipalpis em duas diferentes abordagens: Canarypoxvirus sp. expressando o gene que codifica para a proteína salivar LJM17 e/ou LJL143, e a proteína do intestino médio luloper1 como vacina a proteína salivar LJM17 e/ou LJL143, e a proteína do intestino médio luloper1 como vacina bloqueadora de transmissão". reponame:Repositório Institucional da FIOCRUZ, 2015. https://www.arca.fiocruz.br/handle/icict/12697.
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Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil
As interações entre flebótomo, parasita e hospedeiro desempenham um papel importante na transmissão da leishmaniose. As moléculas provenientes do vetor são relevantes para estas interações e incluem as proteínas da saliva e do intestino médio. Nos flebótomos, as Leishmanias passam por um ciclo de desenvolvimento complexo dentro do intestino médio sob a proteção da matriz peritrófica, necessário para a geração de formas metacíclicas infectantes. As Leishmanias são transmitidas pelos flebótomos que co-injetam parasitas juntamente com a saliva, na derme do hospedeiro. Estudos anteriores demonstraram que a imunização de cães com duas proteínas salivares (LJM17 ou LJL143) de L. longipalpis, resultaram em uma imunidade mediada por células Th1 sistêmica e local afetando a sobrevivência do parasita in vitro. Assim, o objetivo deste trabalho foi avaliar a imunidade conferida pela imunização de cães com DNA e rCanarypoxvirus expressando o gene que codifica para as proteínas salivares de L. longipalpis (LJM17 e/ou LJL143). A imunização com ambas LJL143 e/ou LJM17 induziu uma forte resposta imune humoral. A produção específica do IFN-γ foi observada apenas nas CMSP dos grupos imunizados estimuladas com a proteína. Trinta dias após a última imunização, os cães foram desafiados por via intradérmica com 107 de L. infantum na presença de saliva de L. longipalpis, e a infecção foi detectada no segundo mês após o desafio em todos os grupos. Os cães imunizados com a LJM17 apresentaram maior produção de IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF-α, IP-10 e GM-CSF durante a infecção quando comparados com os controles, indicando que o efeito da imunização induzida por LJM17 persistiu mesmo após o desafio. Adicionalmente, diversos estudos realizados no controle da malária, têm demonstrado o uso de antígenos provenientes do vetor para o desenvolvimento de vacinas bloqueadoras de transmissão. Esta estratégia altruísta de imunização visa criar anticorpos que interfiram no desenvolvimento do parasita no interior do vetor. Assim, na segunda etapa do nosso trabalho, foi testada em cães uma estratégia de imunização utilizando uma proteína extraída do intestino médio do L. longipalpis (Luloper1) para induzir a produção de anticorpos em cães saudáveis e infectados e a interrupção da transmissão no flebótomo. Dessa forma, a imunização de cães saudáveis não infectados ou infectados assintomáticos induziu uma potente produção de anticorpos, porém nenhum efeito de bloqueio de transmissão foi detectado em flebótomos alimentados com o sangue desses animais contendo promastigotas de L. infantum.
Sand fly, parasite, host interactions play an important role in the transmission of leishmaniasis. Vector molecules are relevant for such interactions and include midgut and salivary proteins. In vector sand fly species, Leishmania parasites undergo a complex developmental cycle within the midgut, protected by the peritrophic matrix that is necessary for generation of infectious metacyclics. Leishmania parasites are transmitted by sand flies that co-inject parasites and saliva, in the host’s skin. Previous studies showed immunization of dogs with two proteins (LJM17 or LJL143) from Lutzomyia longipalpis, resulted in a systemic and local Th1 cell-mediated immunity affecting parasite survival in vitro. In this work we evaluated the immunity conferredbyimmunization of dogs with DNA and recombinant Canarypoxvírusexpressing the gene encoding the salivary ofL.longipalpis (LJM17and/orLJL143). Immunization with both LJL143 and LJM17 induced a strong specific humoral response. Specific production of IFN-γ was observed only in protein stimulated PBMC immunized groups. Thirty days after last immunization, dogs were challenged intradermally with 107L. infantum in the presence of L. longipalpis saliva and infection was detected in the second month after challenge in all the groups. It was observed that dogs immunized with LJM17 presented higher IFN-γ, IL-2, IL-6, IL-7, IL-15, IL-18, TNF-α, IP-10 and GM-CSF production during infection when compared with controls, indicating that the effect of immunization induced by LJM17 persisted even after challenge. Adittionally, previous studies, mostly with malaria control, have shown the use of several vector antigens for the development of transmission blocking vaccines. This altruist strategy of immunization aims to raise antibodies that could affect the development of the parasite inside the vector. Thus, in the second stage of our work we tested in dogs an immunization using a protein extracted from L. longipalpis midgut (Luloper1) to evaluate antibodies production in healthy and infected dogs and the interruption of transmission in the sand fly. Immunization of either healthy non infected or asymptomatic infected dogs induced a potent antibodies production, but no blocking transmission effect was detect in sand flies fed with blood of these animals containing L. infantum promastigotes
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Steinkamp, Heidi Marie. "The Role of Macrophages and the Th1 Transcription Factors STAT1 and STAT4 During Visceral Leishmaniasis". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1338335780.
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Eduful, Benjamin Joe. "Synthesis of Novel Agents for the treatment of Infectious and Neurodegenerative diseases". Scholar Commons, 2018. http://scholarcommons.usf.edu/etd/7148.
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Infectious and neurodegenerative diseases continue to be a major concern worldwide. In spite of the great advances in drug therapy for treating various infectious and neurodegenerative diseases, there is still an urgent need for new and improved drugs due to increasing drug resistance among pathogens, emergence of new pathogens, ease of transmission of infections, ineffective available treatments, toxicity associated with current standard of care, aging populations and the lack of better alternative treatment options.
The first part of this manuscript (chapters 1 - 5) describes the synthesis of novel agents active against Leishmania donovani. According to the World Health Organization (WHO), a significant number of deaths worldwide can be attributed to infectious diseases – particularly neglected tropical diseases (NTDs), one of which is leishmaniasis - a complex and clinically diverse disease transmitted through the bite of an infected female phlebotomine sand-fly. The pathogen that causes leishmaniasis develops through a complex life cycle via different morphological changes. Its clinical presentations range from the less severe (cutaneous) to lethal/fatal (visceral) forms depending upon the level of systemic involvement, infecting species and the endemic environment. Treatments (and vaccines) must be species-specific to be particularly effective since sensitivity to commonly used drugs is largely species-specific. Heat shock protein 90 (Hsp 90) has been shown to promote the differentiation of the protozoan parasite that causes leishmaniasis from the promastigote stage to the amastigote pathogenic stages. To this end a series of compounds were prepared based on known Hsp 90 inhibitors, SNX2112 and XL888. The synthetic approach allows the probing of a hydrophobic pocket and rapid access to a collection of anti-leishmanial compounds. The most active compound, was found to be more than twice as active as the climivally used drug, miltefosine, in an infected J774 macrophage at IC50 = 0.65 µM.
The second part of this manuscript (chapters 6 - 9) describes the synthesis novel anti-Alzheimer’s agents. Alzheimer’s disease is a progressive neurodegenerative disease believed to be caused by tau hyperphosphorylation and plaque aggregation in the brain. It is known to affect about 44 million people worldwide and it is marked as the 6th leading cause of death in the United States. Slingshot homology-1 (SSH1) proteins, important protein phosphatases, are promising targets for the discovery of a new generation of small molecule inhibitors as treatment for Alzheimer’s disease, since SSH1 is known to contribute to both tau hyperphosphorylation and plaque aggregation in the brain. Through structure and activity relationships (SAR) studies, two (2) series of compounds were synthesized, thiazoles and pyridones, bearing a carboxylic acid or phosphonic acid functionality as inhibitors of SSH1 enzymes. In the preliminary screening efforts against SSH1 phosphatase activity, the thiazole series were found to be more potent at inhibiting the phosphatase activity than the pyridone series. Among the active thiazole series, eight (8) analogs exhibited significant inhibitory activity over the initial hit compound, observed via phosphatase inhibition curves (using a pNPP phosphatase assay). Further investigations into the molecular target (SSH1) are currently underway.
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Zuque, Maria Angelina da Silva. "Participação de gambás e cães domiciliados como reservatórios de Leishmania infantum e Trypanosoma cruzi georreferenciados nos municípios da Regional de Saúde de Três Lagoas - MS". Botucatu, 2016. http://hdl.handle.net/11449/136267.
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Orientador: Simone Baldini LucheisResumo: A Leishmaniose Visceral e a Doença de Chagas são importantes zoonoses negligenciadas do ponto de vista de saúde pública. Animais domésticos, como o cão, e animais silvestres, como os gambás, fazem parte do ciclo destas zoonoses como fontes de infecção para os vetores. O estudo foi realizado em cinco municípios da Regional de Saúde de Três Lagoas, Mato Grosso do Sul, em 2013 e 2014, com objetivo de identificar a ocorrência da infecção natural por Leishmania spp e Trypanosoma cruzi em gambás (Didelphis albiventris) e cães domiciliados, descrever aspectos epidemiológicos dessas doenças na população canina e humana, seus vetores, e a distribuição espacial da Leishmaniose Visceral Canina usando técnicas do georrefenciamento. Amostras de sangue dos cães foram submetidas a análises sorológicas e moleculares, e a dos gambás somente as análises moleculares. Em relação à pesquisa de anticorpos contra a Leishmania spp., as provas sorológicas de 683 amostras dos cães, revelaram reagentes 320 amostras ao Dual Path Platform, 300 amostras ao Enzyme Linked Immunosorbent Assay e 116 a Reação de Imunofluorescência Indireta. Para Trypanosoma cruzi, pela Reação de Imunofluorescência Indireta, quatro amostras do município de Três Lagoas foram reagentes. Das 683 amostras de sangue total, dos cães submetidas à Reação em Cadeia pela Polimerase, todas foram negativas para Trypanosoma cruzi e obteve-se êxito na amplificação de Leishmania spp em 17 amostras. As 39 amostras de sangue total dos gambás fo... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Visceral Leishmaniasis (VL ) and Chagas disease (CD ) are important neglected zoonoses in accordance with public health. Domestic animals such as dogs and wildlife such as possums , are part of these zoonoses cycles as reservoirs and sources of infection for vectors. The study was conducted in five Regional Health of the municipalities of Três Lagoas , Mato Grosso do Sul in 2013 and 2014 , in order to verify the occurrence of natural infection with Leishmania infantum and Trypanosoma cruzi in opossums (Didelphis albiventris) and the pet dogs, and describe the characteristics of diseases in human and canine population , vectors , and the spatial distribution of canine Visceral Leishmaniasis using techniques georrefenciamento. In dog´s blood there were performed serological analysis and molecular tests ande the samples of the opossums were performed molecular tests. Regarding the research for Leishmania antibodies the techniques serological to 683 dog samples, showed reagent 320 samples to Dual Path Platform, 300 samples to Enzyme Linked Immunosorbent Assay and 116 to Immunofluorescence Antibody Test. For Trypanosoma cruzi, the test Immunofluorescence Antibody Test (IFAT) four samples of Três Lagoas municipality were reactive. Of the samples from dogs 683 submitted to the Polymerase Chain Reaction were all negative by Trypanosoma cruzi gave successful amplification Leishmania spp in 17 of them. The 39 samples of the opossums were all negative by PCR to Leishmania spp. and Trypa... (Complete abstract click electronic access below)
Doutor
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Barbosa, Sofia Diniz de Nazaré. "A leishmaniose canina e os condicionalismos determinados pelas respectivas alterações renais". Bachelor's thesis, Universidade Técnica de Lisboa. Faculdade de Medicina Veterinária, 2011. http://hdl.handle.net/10400.5/3548.
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Dissertação de Mestrado Integrado em Medicina VeterináriaA Leishmaniose é uma doença zoonótica causada pelo protozoário da espécie Leishmania infantum, que existe predominantemente na Bacia do Mediterrâneo. O ciclo de vida deste parasita requer a existência de um vector, um insecto do género Phlebotomus, que é responsável pela transmissão das formas infectantes, os promastigotas metacíclicos, aos hospedeiros vertebrados, nomeadamente os canídeos. Em Portugal, apenas as espécies P. ariasi e P. perniciosus são vectores comprovados de L. infantum. As características clínicas da Leishmaniose canina variam amplamente como consequência dos numerosos mecanismos patogénicos do protozoário, da diversidade de respostas imunológicas desenvolvidas nos hospedeiros e dos diferentes órgãos afectados. A doença renal pode ser a única manifestação clínica nos animais infectados, podendo progredir de proteinúria assintomática até síndrome nefrótico ou Insuficiência Renal Crónica, que é responsável pela degradação do estado geral e a principal causa de morte nos cães com Leishmaniose. A aplicação do tratamento adequado requer, previamente, um diagnóstico precoce da doença e a avaliação do estado sanitário e imunitário do animal, através do exame clínico e de exames complementares, que devem ser repetidos a cada 6 meses. A presença de Insuficiência Renal Crónica impõe algumas limitações no tratamento da Leishmaniose canina, sendo fundamental seleccionar cautelosamente os fármacos e respectivas doses a administrar a cada paciente. Neste estudo retrospectivo foram observados 19 canídeos com Leishmaniose, sendo a sua sintomatologia muito variável, da qual se destaca a linfadenopatia, o emagrecimento e as lesões dermatológicas. As alterações laboratoriais mais frequentes foram a anemia não-regenerativa, a trombocitopénia, a hiperproteinémia com hiperglobulinémia e a proteinúria. O diagnóstico etiológico foi realizado com base nas técnicas de imunocromatografia rápida e de imunofluorescência indirecta. Foi também diagnosticada Insuficiência Renal Crónica em 4 canídeos da amostra. Diferentes fármacos foram utilizados no tratamento etiológico e sintomático, porém foi mais frequente a administração de alopurinol em monoterapia ou em associação com o antimoniato de glucamina.
ABSTRACT - CANINE LEISHMANIASIS AND CONSTRAINTS DETERMINED BY THEIR RENAL CHANGES - Leishmaniasis is a zoonotic disease caused by the protozoan Leishmania infantum, which is the most common species of Leishmania in the Mediterranean basin. The life cycle of this parasite requires the existence of a vector, the insect of the genus Phlebotomus, which is responsible for the transmission of infectious form, the metacyclic promastigotes, to the vertebrate host, namely the dog. In Portugal, only the species P. ariasi and P. perniciosus are proven vectors of L. infantum. The clinical features of canine Leishmaniasis are highly variable as a consequence of the numerous pathogenic mechanisms, the diversity of immune responses of individual hosts and the different organs affected. Renal disease may be the only clinical manifestation in infected dogs and may progress from asymptomatic proteinuria to nephrotic syndrome or to Chronic Kidney Disease, which is responsible for the deterioration of general condition and leading cause of death in dogs with Leishmaniasis. The application of suitable treatment requires, previously, an early diagnosis and assessment of dog’s health and immunity status, by clinical examination and several routine diagnostic tests, which must be repeated every six months. The presence of Chronic Kidney Disease imposes some limitations in the treatment of canine Leishmaniasis and is necessary to select carefully the drugs and doses to be administered in each patient. In this retrospective study, 19 dogs with Leishmaniasis were observed and many clinical signs were found, mainly lymphadenopathy, weight loss and dermatologic lesions. The most frequent laboratory abnormalities were non-regenerative anemia, thrombocytopenia, hyperproteinemia with hyperglobulinemia and proteinuria. The definitive diagnosis was made by a rapid immunomigration test and indirect fluorescent antibody technique. Chronic Kidney Disease was also diagnosed in 4 dogs of sample. Different drugs were used in the etiological and symptomatic treatment, but the administration of alopurinol alone or in combination with glucamine antimoniate were the most frequent.
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Dias, Sueli Meira da Silva. "Estudo bio-molecular de três estoques mistos de trypanosoma cruzi-leishmania spp isolados de pacientes chagásicos crônicos após terapêutica específica para a doença de chagas". Universidade Federal de Goiás, 2006. http://repositorio.bc.ufg.br/tede/handle/tede/3261.
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Thirty cronic chagasic pacients were submitted to specific treatment against Chaga’s disease. The drug of choice was Benznidazole. After treatment laboratorial exams such as parasitological and imunological aiming terapheutic validation were performed. The parasitological analysis is represented by post-treatment hemoculture of 30 cronic chagasic pacients and demonstrated the presence of promastigotes and epimastigotes in 10% (3/30) of pacients, probably characterizing a mixed infection. These hemocultures were named 371, 437 and 438. From these three pacients two were from Goias (GO) and one from Minas Gerais (MG) which are endemic regions to Chaga’s disease as well as to Leishmaniasis which justified the study of this probable co-infection. The work was developed in two phases: in the first phase it was made the biological experimental study of the mixed sotck and in the second phase it was made the confirmation of the Leishmania spp gender through PCR technique. In the experimental biological study the model used was Balb/c isogenic mice and we made an intraperitoneal inoculation of the stocks and we analysed the following parameters: parasitism, hemoculture, sorology by IFI and histopathology. The parasitism observation occured between 48 hours periods through 90 days, and the hemoculture observations occured weekly through 120 days and resulted positive only in mice innoculated with stock 371. The sorology showed anti-T. cruzi antibodies titers in mice innoculated with stocks 371 and 437. The histopathology revealed the presence of amastigotes in tissues cardiac slides from mice innoculated with stock 438, showing that only the epimastigotes forms are present in the mixed stocks and are viable to infect the experimental model used in this work. The confirmation of the Leishmania subgenus envolved in this co-infection was possible through molecular biology techniques. In PCRRFLP, after digestion of PCR products by Hae III restriction enzymes which has specific clivage sites for L. (Viannia) subgender. The samples represented by 371, 437 and 438 stocks and the controls of L. (L.) amazonensis and L. (L.) chagasi did not suffer clivage of its amplified segments in PCR. Only the control constituted by L. (V.) braziliensis suffered clivage resulting in fragments of aproximately 40 and 80 bp confirming undoubtfully that the samples represented by the mixed stocks 371, 437 and 438 isolated from cronic chagasic pacients cantained L. (Leishmania) spp.
Trinta pacientes chagásicos crônicos foram submetidos ao tratamento específico para a doença de Chagas. A droga utilizada foi o Benznidazol. Após o tratamento foram realizados exames laboratoriais compreendendo análises parasitológicas e imunológicas, com a finalidade de validação terapêutica. A análise parasitológica representada pela hemocultura evidenciou a presença concomitante de promastigotas e epimastigotas em 10% (3/30) delas, caracterizando uma provável infecção mista. Essas hemoculturas foram denominadas 371, 437 e 438. Dos três pacientes, dois são procedentes do estado de Goiás (GO) e um do estado de Minas Gerais (MG), regiões endêmicas tanto para a doença de Chagas como para a leishmaniose, o que justificou o estudo desta provável co-infecção. O presente trabalho foi desenvolvido em duas etapas. Na primeira etapa, foi realizado o estudo biológico experimental dos estoques mistos e na segunda, a confirmação do gênero Leishmania spp nos estoques mistos, através da técnica de PCR. No estudo biológico experimental, foram utilizados como modelos camundongos Balb/c isogênicos, realizando-se inoculação intraperitoneal dos estoques. Posteriormente foi analisada a parasitemia, hemocultura, sorologia por IFI e histopatologia. A observação da parasitemia ocorreu a cada 48 horas pelo período de 90 dias, e a hemocultura foi analisada semanalmente por 120 dias, resultando positiva apenas nos camundongos inoculados com o estoque 371. A IFI detectou apenas anticorpos anti-T. cruzi, nos camundongos inoculados com os estoques 371 e 437. A análise histopatológica revelou presença de ninhos de amastigotas nos cortes cardíacos dos camundongos inoculados com o estoque 437, demonstrando no conjunto dessas análises, que apenas as formas epimastigotas presentes nos estoques mistos se mostraram viáveis em infectar o modelo experimental utilizado. A presença de protozoários do gênero Leishmania nos estoques foi confirmada mediante realização de PCR. Na PCR-RFLP, o produto da amplificação foi tratado com a enzima de restrição Hae III que tem sítios específicos de clivagem para o subgênero L. (Viannia). As amostras representadas pelos estoques 371, 437 e 438 e os controles de L. (L.) amazonensis e L. (L.) chagasi não sofreram clivagem de seus segmentos amplificados na PCR. Apenas o controle constituído de L. (V.) braziliensis sofreu clivagem, resultando fragmentos de aproximadamente 40 e 80 pb, confirmando inequivocamente que as amostras representadas pelos estoques mistos 371, 437 e 438 isoladas de pacientes chagásicos crônicos realmente continham L. (Leishmania) spp.
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Kinkead, James Robert H. "Study of the molecular regulation of trypanosomatid phosphofructokinases as drug targets". Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31144.
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The trypanosomatid parasites T. brucei, T. cruzi and Leishmania spp. are responsible for the ‘neglected diseases’ Human African Trypanosomiasis, Chagas disease and Leishmaniasis respectively. In their human infective form in the bloodstream all three trypanosomatid parasites rely heavily on glycolysis for ATP production. Phosphofructokinase (PFK) catalyses the third step of the glycolytic pathway in all organisms using aerobic respiration. It facilitates the phospho transfer from ATP to fructose 6-phosphate (F6P) to make the products fructose 1,6- bisphosphate (F16BP) and ADP. RNAi knockout of T. brucei PFK has shown the enzyme is essential for survival of the bloodstream form parasites. Trypanosomatid PFKs have a unique set of structural and regulatory differences compared to the mammalian host enzyme. These differences, coupled with the availability of trypanosomatid PFK crystal structures present an opportunity for the structure-based design of specific inhibitors against the enzyme. Here we present an enzymatic characterisation of recombinant PFKs from T. brucei, T. cruzi and Leishmania infantum trypanosomatids, their regulation by the allosteric activator AMP, and their inhibition by drug-like inhibitor compounds. Inhibitor compounds (‘CTCB compounds’) were designed against T. brucei PFK with the aim of developing novel treatments against Human African Trypanosomiasis (HAT). We describe the testing, ranking and biophysical characterisation of these compounds as part of a Wellcome Trust Seeding Drug Discovery program. We found that CTCB inhibitor compounds bound to an allosteric pocket unique to trypanosomatid PFKs. We show that the compounds are specific; neither competing with the natural substrates ATP or F6P nor inhibiting the human PFK enzyme. We describe the development and testing of highly potent and specific low molecular weight PFK inhibitors that translate to both killing of cultured T. b. brucei parasites and a cure of stage I HAT in mice models. We describe the tight, 1:1 binding of these compounds with trypanosomatid PFKs, and the thermodynamic characteristics of binding through various biophysical assays. We also show the unprecedented characterisation of the reverse PFK reaction by trypanosomatid and human forms of the enzymes. We found that PFK can also carry out the reverse enzymatic reaction, under physiologically relevant concentrations of ADP and F16BP to produce F6P and ATP. We show that the reverse reaction is also subject to allosteric regulation by AMP, and can be inhibited by the CTCB compounds with a similar potency to the forward reaction. Finally, we describe the mechanism of allosteric activation by AMP and inhibition by the drug-like compounds against trypanosomatid PFKs.
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Dadalto, Carmel Rezende. "Aspectos Doppler e elastográficos renal e esplênico na Leishmaniose visceral canina". Botucatu, 2020. http://hdl.handle.net/11449/192706.
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Orientador: Maria Jaqueline MamprimResumo: O diagnóstico da Leishmaniose visceral é complexo, devido à infinidade de sinais clínicos inespecíficos e por vezes os cães apresentam-se assintomáticos por longos períodos de incubação, contribuindo para a disseminação da doença. Com a finalidade de auxiliar o diagnóstico, o presente estudo tem por objetivo descrever alterações ultrassonográficas ao modo B, Doppler e elastográficos em rim e baço de cães soropositivos para LV. Foram avaliados ao exame ultrassonográfico rins de 33 animais naturalmente infectados por LV, sendo as alterações mais relevantes observadas: o aumento da ecogenicidade cortical (75,75%) e a perda da definição corticomedular (27,27%). O índice de resistividade apresentou-se elevado 0,70 e 0,71, para o rim esquerdo e direito, respectivamente. A dimetilarginina simétrica se mostrou elevada em apenas 12 animais. O escore elastográfico observado com maior frequência foi o dois, referente a tecidos de elasticidade intermediária, tendendo a macio. Também foram avaliados 36 baços de animais soropositivos, o sinal mais frequente foi a heterogeneidade do parênquima (77,77%) com áreas micronodulares hipoecogênicas (60,7%) e esplenomegalia (55,5%). O escore elastográfico esplênico mais observado foi o três, referente a tecidos de elasticidade intermediária tendendo a rígido. As alterações renais e esplênicas descritas no estudo devem ser inclusas como diagnóstico diferencial em cães provenientes de áreas endêmicas.
Abstract: The diagnosis of visceral Leishmaniasis is complex, due to the infinity of nonspecific clinical signs and dogs are often asymptomatic for long incubation periods, which may contribute to the spread of the disease. In order to help early diagnosis, the present study aims to describe sonographic mode B, Doppler and elastographic changes in kidney and spleen of VL seropositive dogs. Kidneys of 33 animals naturally infected with VL were evaluated by ultrasound examination. The most relevant changes were the increase in cortical echogenicity (75.75%) and the loss of corticomedullary definition (27.27%). The resistivity index remained high 0.70 and 0.71 for the left and right kidney respectively. Symmetric dimethylarginine was elevated in only 12 animals. The most frequently observed elastographic score was two, referring to tissues of intermediate elasticity. Thirty-six spleens from seropositive animals were also evaluated, the most frequent sign being parenchymal heterogeneity (77.77%) with hypoechogenic micronodular areas (60.7%), followed by splenomegaly (55.5%), and these changes could appear concomitant or not. The most observed splenic elastographic score was three (47.22%), referring to intermediate elasticity tissues tending to rigid. The renal and splenic changes described in the study should be included as differential diagnosis in dogs from endemic areas.
Doutor
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Jagadesh, Soushieta. "Biogeography of Emerging Infectious Diseases In search for the hotspots of Disease X: A biogeographic approach to mapping the predictive risk of WHO’s Blueprint Priority Diseases Emerging human infectious diseases of aquatic origin: a comparative biogeographic approach using Bayesian spatial modelling Global emergence of Buruli Ulcer Spatial variations between Leishmania species: A biogeographic approach to mapping the distribution of Leishmania species in French Guiana Mapping priority neighborhoods: A novel approach to cluster identification in HIV/AIDS population". Thesis, Guyane, 2020. http://www.theses.fr/2020YANE0007.
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La récente pandémie de Covid19 nous rappelle, si cela était encore nécessaire, que la propagation des maladies infectieuses ignore les frontières géographiques. Les changements combinés de biodiversité locale et l’utilisation des terres, l’augmentation de la connectivité internationale par le transport et le commerce ainsi que la menace imminente du changement climatique a accru le risque d’émergence et de réémergence des maladies infectieuses (EMI). Jusqu’à présent la réponse des politiques de santé publique a été la surveillance passive sans toutefois s’avérer réellement efficace dans la prévention et le contrôle des épidémies. Le choix qui a été fait ici est celui d’une nouvelle approche anticipative, par identification des zones à haut risques d’EMI en se basant sur la détection des facteurs environnementaux les plus favorisant. Parmi ces facteurs on trouve la conversion des terres, la diminution drastique de la biodiversité ou encore le changement climatique. Ainsi la méthode biogéographique a permis d’étudier et d’analyser les EMI à travers différents groupes de taxons de pathogènes comme les bactéries, les virus, les protozoaires et les champignons. L’étude a été portée globalement, ainsi que localement, en Guyane Française, un territoire français d’outre-mer situé en Amérique du Sud. Dans les deux cas, à travers les différents groupes de pathogènes, les risques d’inondation, les récentes conversions de parcelles de forêts en terres agro-minières et l’augmentation du minimum de température due au changement climatique se sont avérés être des facteurs significatifs dans l’émergence globale et locale des maladies infectieuses étudiées. Les principaux résultats de cette thèse sont les suivantes :1. Une approche biogéographique de modélisation de la distribution des EMI en utilisant les bases de données existantes sur les cas cliniques, l’imagerie satellite et un modèle statistique non conventionnel est efficace pour détecter précocement les régions à risque, permettre d’améliorer la prévention, et contrôler leur diffusion.2. Il est possible d’anticiper les EMI en identifiant et en gérant précocement les facteurs favorisant ayant un lien direct avec l’anthropisation de l’environnementThe COVID-19 pandemic highlights that the spread of infectious diseases goes beyond geographical boundaries. Simultaneous changes in local biodiversity and land use, the increasing international connectivity through human transport and trade and the imminent threat of climate change have increased the risk of the emergence and reemergence of infectious diseases. The current public health response to emerging infectious diseases (EID) by passive surveillance has proven largely ineffective in preventing and controlling disease outbreaks. The way toward is to “get ahead of the curve” by identifying potential hotspots of disease emergence and detecting the environmental triggers such as land transformation, biodiversity loss and climate change. I used a biogeographic approach to study and analyze disease emergence across different taxonomic pathogen groups such as bacterial, viral, protozoal and fungal, globally and in French Guiana, a French Overseas territory located in South America. I found that regions at risk of floods, recent conversion of forest to agricultural lands and increasing minimum temperature (i.e. temperature at night) caused by cli mate change were drivers for disease emergence locally and globally across the different pathogen groups. The main findings of the PhD thesis are the following:1. Biogeographic approach to mapping the distribution of EIDs with using existing human cases data, remote sensing imagery and unconventional statistical models is effective to “get ahead of the curve” in the detection of regions at risk and the management of EIDs.2. EIDs are not unprecedented but predictable by identifying and managing the triggers of disease emergence, which have a direct link with the anthropization of the environment
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Piedra, Ysabel Catalina Montoya. "Molecular analysis of antigen genes in Peruvian Leishmania". Thesis, University of Cambridge, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.309146.
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Gushi, Letícia Tsieme [UNESP]. "Dinâmica populacional de minicírculos de cinetoplastos em Leishmania infantum chagasi". Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/102852.
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Leishmaniose Visceral Americana (LVA) é uma doença tropical negligenciada em expansão no Brasil, ocorrendo em áreas onde antes não havia registro. Seu agente etiológico e a Leishmania chagasi um protozoário pertencente à classe Kinetoplastida caracterizada por uma organela denominada cinetoplasto a qual possui um DNA organizado em uma rede contendo maxicírculos, responsáveis pelas funções respiratórias e minicírculos, envolvidos na pordução de RNAs-guia, os quais possuem um papel importante na edição dos RNAs dos maxicírculos. Os minicírculos são divididos em uma região convervada de aproximadamente 120 p.b. e uma região variável de aproximadamente 600 p.b. O foco desse estudo está na análise das seqüências da região variável a fim de entender sua distribuição nos diferentes estágios de vida da L. chagasi. As amostras foram coletadas de cães e pacientes sintomáticos por aspiração dos linfonodos e, em alguns casos, foram obtidas culturas primárias. A extração do DNA foi realizada com o kit comercial Nucleo Spin Blood Kit (Macherey - Nagel) seguindo as instruções do protocolo. O kDNA foi amplificado por PCR, utilizando o par de oligonucleotídeos LIN R4 - forward (5’-GGT TGG TGT AAA ATA GGG-3) e LIN 19 - reverse (5’-GAA CGC CCC TAC CCG-3’), produzindo um fragmento de aproximadamente 720 p.b. Os produtos da PCR foram clonados no vetor pTZ57R/T de acordo com o protocolo do InsTAclone PCR cloning kit. As seqüências (aproximadamente 182) foram individualmente comparadas com as depositadas no GenBank, alinhadas com o software Clustal X2 e tiveram uma árvore filogenética construída utilizando o software MEGA 4.0 adotando o algoritmo UPGMA e escolhendo um bootstrap com 1000 replicatas. A distribuição entre os diferentes hospedeiros foi homogênea. A princípio, um lato polimorfismo é observado, mas...
American Visceral Leishmaniasis (AVL) is a neglected tropical disease in expansion in Brazil currently occurring in areas where there has never been reports. Its etiologic agent is Leishmania chagasi, a protozoan belonging to the order Kinetoplastida characterized by an organelle named kinetoplast wich has a DNA organized in a network containing maxicircles, responsible for respiratory functions and minicircles, involved in the production of guide RNAs, which play a role in the RNA editing of maxicircles. The minicircles are divided into an approximately 120 b.p. conserved region and an approximately 600 b.p. variable region. The focus of this study is on the sequence analysis of the minicircles variable region in order to understand its distribution on different life stages of L. chagasi. Samples were collected from dogs and symptomatic patients by lymphonod aspiration and, in some cases, primary cultures were obtained. DNA extraction was carried out with the commercial kit Nucleo Spin Blood Kit (Macherey - Nagel) following its protocol. kDNA was amplified by PCR, using a pair of oligonucleotides LIN R4 - forward (5’-GGT TGG TGT AAA ATA GGG-3) and LIN 19 - reverse (5’-GAA CGC CCC TAC CCG-3’), producing a fragment of 720 b.p. PCR products were cloned in pTZ57R/T vector according to the InsTAclone PCR cloning kit protocol. Sequences (182) were individually compared with the ones deposited at the GENBANK, aligned with Clustal X2 software and had a phylogenetic tree constructed utilizing MEGA 4.0 software adopting UPGMA algorithm and choosing bootstrap with 1000 replicates. Sequences distribution among different hosts was homogeneous. At first, high polymorphism is observed but, when analyzed in more detail, i.e. by branch, sequences proved to be conserved and minimal SNP (Single Nucleotide Polymorphism) was found... (Complete abstract click electronic access below)
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Gushi, Letícia Tsieme. "Dinâmica populacional de minicírculos de cinetoplastos em Leishmania infantum chagasi /". Botucatu, 2012. http://hdl.handle.net/11449/102852.
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Orientador: Paulo Eduardo Martins RibollaBanca: Hiro Goto
Banca: Jayme Augusto de Souza Neto
Banca: Cassiano Victória
Banca: Carlos Magno Castelo Branco Fortaleza
Resumo: Leishmaniose Visceral Americana (LVA) é uma doença tropical negligenciada em expansão no Brasil, ocorrendo em áreas onde antes não havia registro. Seu agente etiológico e a Leishmania chagasi um protozoário pertencente à classe Kinetoplastida caracterizada por uma organela denominada cinetoplasto a qual possui um DNA organizado em uma rede contendo maxicírculos, responsáveis pelas funções respiratórias e minicírculos, envolvidos na pordução de RNAs-guia, os quais possuem um papel importante na edição dos RNAs dos maxicírculos. Os minicírculos são divididos em uma região convervada de aproximadamente 120 p.b. e uma região variável de aproximadamente 600 p.b. O foco desse estudo está na análise das seqüências da região variável a fim de entender sua distribuição nos diferentes estágios de vida da L. chagasi. As amostras foram coletadas de cães e pacientes sintomáticos por aspiração dos linfonodos e, em alguns casos, foram obtidas culturas primárias. A extração do DNA foi realizada com o kit comercial Nucleo Spin Blood Kit (Macherey - Nagel) seguindo as instruções do protocolo. O kDNA foi amplificado por PCR, utilizando o par de oligonucleotídeos LIN R4 - forward (5'-GGT TGG TGT AAA ATA GGG-3) e LIN 19 - reverse (5'-GAA CGC CCC TAC CCG-3'), produzindo um fragmento de aproximadamente 720 p.b. Os produtos da PCR foram clonados no vetor pTZ57R/T de acordo com o protocolo do InsTAclone PCR cloning kit. As seqüências (aproximadamente 182) foram individualmente comparadas com as depositadas no GenBank, alinhadas com o software Clustal X2 e tiveram uma árvore filogenética construída utilizando o software MEGA 4.0 adotando o algoritmo UPGMA e escolhendo um bootstrap com 1000 replicatas. A distribuição entre os diferentes hospedeiros foi homogênea. A princípio, um lato polimorfismo é observado, mas... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: American Visceral Leishmaniasis (AVL) is a neglected tropical disease in expansion in Brazil currently occurring in areas where there has never been reports. Its etiologic agent is Leishmania chagasi, a protozoan belonging to the order Kinetoplastida characterized by an organelle named kinetoplast wich has a DNA organized in a network containing maxicircles, responsible for respiratory functions and minicircles, involved in the production of guide RNAs, which play a role in the RNA editing of maxicircles. The minicircles are divided into an approximately 120 b.p. conserved region and an approximately 600 b.p. variable region. The focus of this study is on the sequence analysis of the minicircles variable region in order to understand its distribution on different life stages of L. chagasi. Samples were collected from dogs and symptomatic patients by lymphonod aspiration and, in some cases, primary cultures were obtained. DNA extraction was carried out with the commercial kit Nucleo Spin Blood Kit (Macherey - Nagel) following its protocol. kDNA was amplified by PCR, using a pair of oligonucleotides LIN R4 - forward (5'-GGT TGG TGT AAA ATA GGG-3) and LIN 19 - reverse (5'-GAA CGC CCC TAC CCG-3'), producing a fragment of 720 b.p. PCR products were cloned in pTZ57R/T vector according to the InsTAclone PCR cloning kit protocol. Sequences (182) were individually compared with the ones deposited at the GENBANK, aligned with Clustal X2 software and had a phylogenetic tree constructed utilizing MEGA 4.0 software adopting UPGMA algorithm and choosing bootstrap with 1000 replicates. Sequences distribution among different hosts was homogeneous. At first, high polymorphism is observed but, when analyzed in more detail, i.e. by branch, sequences proved to be conserved and minimal SNP (Single Nucleotide Polymorphism) was found... (Complete abstract click electronic access below)
Doutor
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Toepp, Angela Jean. "Host factors that alter Leishmania infantum transmission". Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6313.
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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.
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Smith, Helen Katherine. "Combinatorial chemistry and polyamines in the battle against trypanosomes and leishmanias". Thesis, University of Southampton, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.242499.
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Petri, Simone Carolina Soares. "Efeitos de derivados nitro heterocíclicos sintéticos sobre formas promastigotas e amastigotas intracelular de Leishmania (Leishmania) infantum". Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-26102015-145221/.
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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 visceralINTRODUCTION: 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
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Federico, Stefano. "Towards innovative tools against vector-borne diseases: focusing on Plasmodium and Leishmania spp". Doctoral thesis, Università di Siena, 2022. http://hdl.handle.net/11365/1194525.
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Up to date, the World Health Organization (WHO) recognizes twenty conditions belonging to neglected tropical diseases (NTDs) caused by parasites, viruses, bacteria, and snake envenoming that affect some of the World’s poorest areas, predominantly in Africa, Asia, and the Americas. NTDs, that affect more than a billion people worldwide, are referred to as “neglected” as they receive inadequate attention, e.g., in terms of research funding, when compared to other diseases. Of the twenty NTDs recognized by the WHO, twelve are caused by parasites. Based on data provided by the 2019 Global Burden of Disease Study (GBD), over 20 million disability adjusted life years (DALYs) are caused by NTDs and approximately 750,000 people died because of NTDs and malaria. Taken together, these data lead malaria and NTDs to be the 15th leading cause of death worldwide.
Regarding malaria, based on our previous study on bridged bicyclic 2,3-dioxabicyclo[3,3,1]dioxanes as antimalarial agents, in this work we aimed at improving the potency and the pharmacokinetic profiles of the latter by developing two new classes of bridged bicyclic endoperoxides. The introduction of protonable chains at R1 led to a marked increase in potency with respect to previous derivatives; additionally, the introduction of until-now unexplored triazine-based R1 substituents paved the way for the rational design of novel optimized antimalarial agents. Both classes of endoperoxides showed good inhibitory potency toward P. falciparum, and these results were also rationalized by in silico analysis of the interaction between the peroxide bridge and Fe(II)-heme. Furthermore, taking inspiration from the anticancer properties of ART-derived dimers, three new sets of endoperoxide-based dimers were designed and synthesized. The study design aimed at unveiling the main feature required for the explication of the antitumor activity. Preliminary biological investigation performed in human leukemia HL-60 cell line highlighted compounds 66d and 66g as the most promising derivatives of the series. In conclusion, 24 new chemical entities were synthesized and subjected to biological investigation.
As per NTDs, we have identified 25 new chemical entities active against Leishmania (and possibly other trypanosomatids) trypanothione reductase, derived from the hit compound 138a. The potent and selective TR inhibitor 138a, identified by screening of GSK LeishBox, acts by selectively bind the TS2 binding pocket of TR (with respect to hGR). Further structural information were obtained by crystallography studies, which led to the resolution of the co-crystal structure of 138a in complex with TbTR, thus confirming the mechanism of inhibition. The intensive SAR analysis led to the identification of the most important features of the parent compound. The most promising derivatives, in terms of IC50 values against LiTR, were also evaluated in phenotypic assays against axenic amastigote and microphage-infecting promastigote life cycle stages of L. infantum. Moreover, the toxicity profile for some of the best compound was assessed in 3T3 and HepG2 cell lines to get preliminary information about the selectivity of the latter versus human hosts. Further biological studies are ongoing to validate the therapeutic potential of this new class of TR inhibitors in an in vivo murine model of Leishmania infection.
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Franco, Fernando Alves de Lima. "Caracterização da região genômica META 1 de Leishmania (Leishmania) amazonensis e comparação com a região ortóloga de L. (L.) major". Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-03122008-102437/.
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A caracterização de sequências codificadoras presentes nas vizinhanças do gene META 1 permitiu a identificação de alguns genes expressos preferencialmente em estágios infectivos de L. (L.) amazonensis. Um dos genes presentes é regulado de forma distinta, observando-se maior abundância do RNA em formas amastigotas. Este gene foi denominado LaLRR17 por codificar uma proteína contendo em sua região central 6 repetições ricas em leucina (LRR). As LRR são motivos presentes em diversas famílias de proteínas e são responsáveis pela formação de uma estrutura capaz de estabelecer interações protéicas. A região central da proteína LRR17 apresentou similaridade com a porção carboxi-terminal da proteína NOD 3 humana. A proteína LRR17 foi localizada no citosol de macrófagos infectados com L. (L.) amazonensis. Para caracterizar a função da proteína LRR17 foram obtidas linhagens de L. (L.) amazonensis expressoras do gene LmjLRR17. Essas linhagens mutantes foram mais infectivas em macrófagos in vitro quando comparadas com a linhagem selvagem. Avaliamos também o papel das proteínas NOD 1 e NOD 2 na infecção por L. (L.) amazonensis e L. (L.) major para estabelecer a possível relação da proteína LRR17 na interação com essas vias de defesa celular do macrófago.The characterization of coding sequences in the vicinity of the META 1 gene allowed the identification of some genes preferentially expressed in L. (L.) amazonensis infective stages. One of the identified transcripts presents a distinct pattern of expression with higher levels of mRNA in amastigotes. This gene was named LaLRR17 since it encodes a 72 kDa protein with 6 leucine-rich repeats (LRR) in its central region. Leucine-rich repeats (LRR) are present in several families of proteins and are responsible for the formation of a structure capable of establishing protein interactions. The central region of the LRR17 protein showed similarity with the carboxyl-terminal portion of the NOD 3 human protein. The LaLRR17 protein is secreted to the cytoplasm of L. (L.) amazonensis-infected macrophages. To characterize the function of the LRR17 protein we obtained strains of L. (L.) amazonensis expressing the LmjLRR17 gene. These mutant strains were more infective to macrophages in vitro when compared with the wild type strain. We also evaluated the role of NOD 1 and NOD 2 proteins in infections with L. (L.) amazonensis and L. (L.) major to investigate a possible role of the LRR17 protein in the interaction with these defense pathways in macrophages.
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Elnaiem, Dia-Eldin Ahmed. "Oviposition of Lutzomyia longipalpis (Diptera: Psychodidae) and development of Leishmania chagasi (Kinetoplastida: Trypanosomatidae) in the vector". Thesis, University of Liverpool, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316732.
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Luba, James. "Studies of Leishmania major Pteridine Reductase 1, a Novel Short Chain Dehydrogenase". eScholarship@UMMS, 1997. https://escholarship.umassmed.edu/gsbs_diss/45.
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Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis.
PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates.
The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed.
The stereochemistry of hydride transfer
The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family.
The kinetic mechanism of PTR1
When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic.
Catalytic Residues of PTR1
The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF.
Crystallization of PTR1 and PTR1-ligand complexes
The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.
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Cheleski, Juliana. "Planejamento de inibidores da enzima diidroorotato desidrogenase de Trypanosoma cruzi por biocalorimetria". Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/75/75131/tde-19052011-110337/.
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A doença de Chagas, causada pelo protozoário flagelado Trypanosoma cruzi, é uma doença tropical que enseja morte/morbidade de milhões de pessoas na América Latina. Por processos migratórios, vem-se estendendo ao sul dos Estados Unidos, Canadá, Europa, Austrália e Japão. Essa doença tem sido considerada super-negligenciada pela indústria farmacêutica, já que os dois fármacos disponíveis para o seu tratamento foram introduzidos há mais de quarenta anos e apresentam baixa eficácia com vários efeitos colaterais severos. Mais recentemente, a Organização Mundial da Saúde considerou a doença de Chagas, dentre outras, como a doença da pobreza! Com esse cenário completamente desfavorável aos portadores da doença, é necessária a descoberta, desenvolvimento e introdução de novos fármacos para o tratamento eficiente e seguro da doença de Chagas. Dentro desse contexto, este trabalho representa uma importante contribuição para o entendimento das razões moleculares da ação farmacológica de substâncias químicas bioativas de interesse à farmacoterapia da doença de Chagas. Ao nível molecular, a enzima pertencente à via de síntese de novo de nucleotídeos de pirimidinas, diidroorotato desidrogenase do Trypanosoma cruzi (TcDHODH), é um alvo promissor para a descoberta e desenvolvimento de candidatos a fármacos de interesse para o tratamento da doença de Chagas.
Os conceitos e ferramentas da química medicinal computacional, tais como os ensaios virtuais in silico, foram usados para a identificação de inibidores da TcDHODH. Vinte e seis substâncias inéditas como inibidores da TcDHODH foram adquiridos comercialmente e avaliados experimentalmente através da Calorimetria de Titulação Isotérmica (ITC) para a determinação do mecanismo de inibição e da constante cinética de afinidade (Kiapp).
Na etapa de docagem molecular, o objetivo era identificar moléculas que apresentassem uma boa afinidade pelo sítio ativo da enzima TcDHODH. A primeira série de ligantes selecionados dos métodos in silico, apresentou inibição enzimática na concentração de micromolar com eficiência média de ligante de 0,50 kcal mol-1 átomo-1. Devido à baixa massa molecular (aproximadamente 200 kDa) e a alta eficiência de ligante, essa série foi considerada como constituída de excelentes substâncias com elevado poder de reconhecimento biomolecular. Por isso, foram caracterizadas como substâncias passíveis de otimização no processo do-ligante-para-substância matriz.
As enzimas TcDHODH e DHODH de Leishmania major (LmDHODH) têm sítios ativos com elevado grau de similaridade. Portanto, usando a enzima LmDHODH como padrão de substituição da TcDHODH é possível fazer a descrição do modo de interação do co-complexo TcDHODH-inibidor. O modo de ação descrito através da resolução da estrutura cristalográfica de raios-X, além de validar ortogonalmente os resultados cinéticos obtidos por ITC - que identificou as substâncias como inibidores competitivos (por interação direta no sítio ativo da enzima TcDHODH), geraram hipóteses farmacofóricas para a busca de novas moléculas (chamadas de segunda geração), agora com padrão superior de reconhecimento molecular do sítio da TcDHODH. Para validar complementarmente a hipótese, foi demonstrado que os inibidores da TcDHODH inibem, similarmente, a LmDHODH.
Uma análise cuidadosa da estrutura tridimensional da enzima TcDHODH, demostrou a possibilidade de ocupação do sítio S2 que se estende além da região do sítio catalítico S1, permitindo assim o aumento da afinidade biomolecular com os inibidores. Além disso, o sítio S2 não é encontrado na estrutura da proteína de humanos (HsDHODH), podendo ser uma região passível de seletividade frente à enzima TcDHODH.
O emprego adequado dessa hipótese resultou na otimização dos ligantes identificados previamente para substâncias mais potentes que inibiram a enzima de forma competitiva em relação ao substrato diidroorotato (DHO) em valores Kiapp de 121 ± 14 nM e 190 ± 10 nM.
A técnica de ITC foi fundamental no processo de descoberta de inibidores enzimáticos, pois se mostrou extremamente susceptível à determinação da interação intermolecular enzima-inibidor, permitindo acompanhar a cinética da reação e obter os valores da constante de afinidade de maneira precisa e acurada. Com isso, a taxa de acerto obtida nesta tese foi de 46%, considerando-se apenas as substâncias com valores de Ki app < 100 µM. Esse é um número favoravelmente apreciável, já que na literatura ele gira em torno de 1-10% quando o planejamento in silico é realizado, quando comparado às taxas de acerto dos métodos de ensaio em larga escala (HTS), entre 0-2 %, os resultados alcançados neste trabalho são ainda mais significativos.
Além disso, as substâncias químicas selecionadas através da integração de métodos in silico e biocalorimétricos apresentam elevado grau de complexidade no processo biomolecular de interação enzima-ligante, que permite classificá-las para as fases seguintes da gênese planejada de fármacos.
American trypanosomiasis or Chagas disease, caused by the haemoflagellate Trypanosoma cruzi, is a tropical disease that affects millions of people in Latin America. Epidemiology of Chagas disease in non-endemic countries is attained by immigration as the disease also affects people in the United States, Canada, Europe, Australia and Japan. However, the United States are not to be written off as an area of nonendemicity for Chagas disease like Europe or Asia because the southern states have enzootic T. cruzi transmission that involves triatomine species and hosts such as raccoons, opossums, and domestic dogs. Even though, this disease has been considered as a super-neglected from the big Pharma Industry viewpoint since the only available drugs for its treatment were introduced in the market more than forty years ago and worsen is that they have low efficacy and cause various severe side effects.
Although the current clinical scenario is of course discouraging and is far from being even a soothing treatment for those who suffer from the disease, it prompt ones to set efforts towards the need of discovering and developing new efficacious and safe drugs to treat Chagas disease.
Our research group covers the concept of enzymes acting as targets for the action of drugs. Once T. cruzi has many druggable targets, the dihydroorotate dehydrogenase enzyme (TcDHODH) that belongs to the de novo pyrimidine nucleotide synthetic pathway has been chosen for the search of new inhibitors that may be of use in the treatment of Chagas disease. To accomplish with this and considering that inhibitors are molecules that decrease enzyme activity leading to parasite death, we used the concepts and tools of modern computational medicinal chemistry such as in silico screening of small molecules that bind to the active site of the TcDHODH.
After a thoroughly program of virtually screening thousands of compounds, 26 were purchased from commercially available sources and experimentally assayed against the TcDHODH using Isothermal Titration Calorimetry (ITC) in order to determine the mechanism of inhibition and the kinetic affinity constant (Kiapp).
The first series of inhibitors selected from our in silico strategy were evaluated by ITC to yield compounds that inhibited the TcDHODH in the micromolar concentration range with an average of 0.50 kcal mol-1 atom-1 ligand efficiency (LE). Because the assayed compounds have low molecular weight (ca. 200 kDa) and high LE, which bring them to the specific bimolecular pattern recognition all of them were considered good inhibitors capable of being selected to enter the hit-to-lead optimization process.
The detailed description of the ligand-enzyme mode of binding (MOB) is thoroughly accomplished by solving the X ray crystal structure of the surrogate Leishmania major DHODH enzyme (LmDHODH), which has a high degree of similarity with the enzyme TcDHODH. The MOB credited to be in the active site of the TcDHODH orthogonally validated the ITC kinetic experimental data obtained for all ligands as competitive inhibitors that interact at the active site of the TcDHODH and helped to generate pharmacophoric hypotheses for the search of new second generation molecules acting against the enzyme TcDHODH. Analyzing the 3D structure of the TcDHODH along with its surrogate LmDHODH, we envisaged the possibility of compounds to extend their side chain beyond the region of the catalytic site (called S1), and interacting in a region called S2, so to increase binding affinity. Moreover, the TcDHODH S2 site that is not found in the 3D protein structure of humans (HsDHODH) is likely to offer new insights for the search of inhibitors whose binding to this S2 site can pave the roads towards the needed structural basis for selective inhibition of TcDHODH.
The most potent compounds inhibited the enzyme competitively with respect to the substrate dihydroorotate (DHO) at Kiapp values of 121 ± 14 nM and 190 ± 10 nM, which constitutes high affinity TcDHODH inhibitors. The ITC technique was pivotal to this process of enzyme inhibitors discovery, because it proved to be extremely sensitive thus allowing to monitor the kinetics of the reaction and to obtain precise and accurate values of affinity constants.
The hit rate obtained in this work, considering only those compounds with Kiapp < 100 µM, was 46%. This is a really high number, since literature values range from 1 to 10% when the planning new inhibitors via in silico methods when compared to the success rates obtained by the methods of testing on large scales (HTS), 0-2 %, the results achieved in this work are even more significant. Moreover, the compounds selected through the integration of in silico and calorimetric methods showed a high degree of complexity in the process of bimolecular enzyme-ligand recognition, which allows to pass them to the next phase of the drug design process.
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LOURENCO, CECILIA de O. "Avaliacao da atividade de diferentes venenos de serpentes, nativos ou irradiados, com radiacao gama de sup(60)Co, quanto ao poder inibitorio do crescimento de Leishmania (Leishmania) amazonensis". reponame:Repositório Institucional do IPEN, 2000. http://repositorio.ipen.br:8080/xmlui/handle/123456789/10875.
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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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Ma, Wai Sheung. "Natural Product Drug Discovery against Tropical Diseases". Scholar Commons, 2011. http://scholarcommons.usf.edu/etd/3224.
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This dissertation describes the isolation of secondary metabolites from natural origins through a series of chromatographic techniques and spectrometric characterization in the effort of drug discovery. The isolated compounds obtained were used as drug leads against tropical diseases, namely malaria and leishmaniasis. While first chapter offers an introduction on the use of a natural product by itself as an effective therapeutic and its role on inspiring the discovery of new drugs, the later chapters will concentrate on isolation and characterization of bioactive natural products from an Antarctic sponge and mangrove endophytic fungi during the dissertation work.
The second chapter describes the attempt to develop a new method of solving the absolute configuration of tertiary alcohol using lanthanide chiral shift reagent and 13C NMR spectroscopy. The third chapter describes the isolation of five new steroids, norselic acids A-E, from Crella sp. collected in Antarctica. The structures of the norselic acids were established by NMR and MS techniques. The absolute stereochemistry of norselic acid A was elucidated by SXRD. The antimicrobial and anti-leishmania activities of norselic acid A have been studied. Norselic acid A displays antimicrobial activities against methicillin-resistant S. aureus (MRSA), S. aureus, E. faecium, and C. albicans. Norselic acids B-E exhibit mild antimicrobial activities. All norselic acids exhibit strong cytotoxicity against leishmania.
The fourth and fifth chapters describe a Medicine for Malaria Venture (MMV) funded malaria bioassay-guided screening program. The chemical investigation of the crude endophytic fungal extracts has led to the isolations of a series of known cytochalasins along with the discovery of a few new compounds, including a new simple carboxylic acid, and several known and novel compounds belonging to the dimeric xanthone family. Majority of the cytochalasins display mild cytotoxicity and outstanding inhibition to chloroquine-resistant reference strain Plasmodium falciparum (W2) with IC50 ranging from 25.8 nM to 2900nM. However, their cytostatic properties hinder them from being a good drug candidate. The dicerandrols display good activity with the lowest IC50=0.63 μM against malaria with low cytotoxicity. The structures of the compounds isolated and the associated anti-malarial activities are reported herein.
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BORBOREMA, SAMANTA E. T. "Biodistribuição do antimoniato de meglumina em animais sadios e infectados com Leishmania (L.) chagasi". reponame:Repositório Institucional do IPEN, 2005. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11341.
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IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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Wheeler, Richard John. "Generation, regulation and function of morphology in Leishmania and Trypanosoma". Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:c44354bc-5a93-4fce-a716-bb0a63131901.
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Little is known about the generation of Leishmania morphology and the function of morphology in trypanosomatids, despite every species having characteristic cell shapes and undergoing changes in morphology between life cycle stages. To address this I analysed morphogenesis of the cell body and flagellum through the cell cycle of the Leishmania insect (promastigote) life cycle stage using a novel method for determining cell cycle stage from cell size and DNA content. This showed cell body morphology is generated by growth and then remodelling of cell shape around mitosis and cytokinesis. Mathematical modelling of flagellum growth indicated flagellum length continues to increase over multiple cell cycles and does not reach a defined length. I also observed little link between the cell cycle and flagellum length regulation during differentiation to the mammalian macrophage-inhabiting (amastigote) life cycle stage. Analysis of motility showed the diverse flagellar lengths of promastigote Leishmania cells bestow different swimming abilities, and the capacity of Leishmania promastigotes for highly directional swimming differs sharply from trypomastigote Trypanosoma brucei. This difference did not arise from altered flagellar beating therefore appeared to be linked to morphology. Together these indicate the mechanisms of cell body morphogenesis, flagellum length regulation, life cycle stage differentiation and the swimming abilities of the cells the morphogenetic processes generate differ significantly between Leishmania and T. brucei. These insights motivated the programming of automated micrograph analysis tools based on a new DNA staining method to support similar future morphometric analyses. This is the first comprehensive comparison of morphogenesis and function of morphology in a promastigote and a trypomastigote and, by considering these new insights in the context of existing molecular biology and the morphological diversity across many trypanosomatid species, give insight into basic Leishmania biology, the shared molecular mechanisms underlying morphogenesis and the potential functions of the diverse morphologies which are seen in different trypanosomatid species and life cycle stages.
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BONETTI, FRANCO C. "Estudo do uso da radiação ionizante como ferramenta de seleção de formas promastigotas metacíclicas de Leishmania amazonensis, e a indução de resposta imunológica em modelos experimentais". reponame:Repositório Institucional do IPEN, 2006. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11548.
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Tese (Doutoramento)
IPEN/T
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP
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Soutter, Francesca. "Canine leishmaniosis : immunogenetics of response to infection and vaccination". Thesis, Royal Veterinary College (University of London), 2016. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.701667.
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Bonetti, Franco Claudio. "Estudo do uso da radiação ionizante como ferramenta de seleção de formas promastigotas metacíclicas de Leishmania amazonensis e a indução de resposta imunológica em modelos experimentais". Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/85/85131/tde-16042012-105125/.
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Atualmente, milhões de pessoas, por todo o globo, estão sob risco de serem infectados por um protozoário transmitido vetorialmente por pequenos insetos flebotomíneos. Este parasita é a Leishmania spp., causadora de uma patologia de amplo espectro, que varia desde a moléstia cutânea (tegumentar) até a visceral (kala-azar). A leishmaniose cutânea é a manifestação clínica de maior ocorrência (mais de 90% dos casos). A radiação ionizante, gerada em fonte de 60Co, tem sido utilizada com sucesso para promover alterações físico-químicas em diferentes protozoários, incluindo a Leishmania spp. Em trabalhos anteriores determinou-se que formas promastigotas de Leishmania amazonensis, irradiadas com diferentes doses de radiação gama, perdem sua viabilidade mantendo, porém, sua imunogenicidade. No presente trabalho, estudouse a utilização da radiação ionizante como ferramenta na seleção de formas metacíclicas do parasita em cultura axênica para a possível produção de imunógenos irradiados mais eficientes. Os resultados demonstram que culturas irradiadas com 400 Gy de radiação gama, possuem uma concentração de aproximadamente 75% de parasitas metacíclicos, capazes de produzir, in vitro, uma infecção que mimetiza a ocorrida naturalmente. Estes parasitas irradiados têm sua estrutura celular interna modificada mantendo, entretanto, seu arcabouço externo intacto. Camundongos de uma linhagem suscetível imunizados com leishmanias irradiadas com diferentes doses tiveram sua produção de imunoglobulinas aumentada, e mantiveram os títulos elevados após o desafio com parasitas não irradiados. Em outras linhagens pesquisadas este padrão se manteve, porém em títulos menores, sendo que camundongos imunodeficientes não responderam à imunização nem ao desafio.Actually, millions of people around the globe are under the risk of infection by a protozoan transmitted by a bit of a sand fly. This parasite is a Leishmania spp. This causes a wide spectrum disease, since a coetaneous disease to a visceral one. The coetaneous form is the major clinical manifestation (above 90%). The ionizing radiation, produced in a 60Co font, had being successes used to promote physical-chemical transformations on different protozoans, including Leishmania spp. In previous work was determined that promastigotes forms of Leishmania amazonensis, irradiated with different doses of radiation, lost their viability maintaining, however, their immunogenicity. In this work, was studied the use of ionizing radiation as a tool for selection of metaciclic forms of the parasite in axenic culture, for a possible efficient irradiated immunogen production. Our results shown that cultures irradiated with 400 Gy of gamma irradiation, has 75% of metaciclic form, which are capable to produce, in vitro, an infection that is similar the natural occurrence. These irradiated parasites have their internal cellular structure modified, maintaining their external structure intact. Susceptible strain of mice immunized with leishmania irradiated with different doses had high immunoglobulin production, and maintained this production after the challenge with naive parasites. In other strains this default was similar, however in lower titles. Immunodeficient mice didnt produce immunoglobulin nor on the immunization or on the challenge.
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Fiebig, Michael. "Characterisation of the transcriptomes of Leishmania mexicana promastigotes and amastigotes". Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:ff1a5033-e1a4-40cc-8204-7de904ba7aa2.
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Leishmania spp. undergo substantial adaptations from being promastigotes, found in sandflies, to being amastigotes, residing in parasitophorous vacuoles within mammalian macrophages. In the past, microarray studies have sought to elucidate these adaptations using axenic amastigote systems or amastigotes purified from host-cells, raising the question whether the observed transcriptomic signatures were a true reflection of intracellular amastigotes. Moreover, with ever-improving genome annotations being available, it is clear that these studies failed to address the transcriptomic behaviour of a considerable number of transcripts. In the work presented herein, I employed RNA-sequencing to obtain transcriptomic profiles of Leishmania mexicana axenic promastigotes (PRO), axenic amastigotes (AXA) and intracellular amastigotes (AMA) in murine bone-marrow derived macrophages. The intracellular amastigotes were not purified from host cells, but instead sequencing reads assigned to a hybrid L. mexicana - Mus musculus genome and the transcriptomes separated in silico. We were able to map pre-mRNA processing sites, thereby defining transcript boundaries, proposing 184 truncations and 1253 extensions of existing gene models as well as discovering 936 novel genes. Mass-spectrometric evidence was obtained for both proposed extended and novel proteins. Using this improved genome annotation, we generated gene expression profiles for AMA, AXA and PRO, identifying 3832 differentially expressed transcripts between PRO and AMA as well as 2176 between PRO and AXA and 1234 between AXA and AMA. Transcripts differentially expressed between AMA and PRO correlated well with previous reports, were enriched for novel transcripts identified in this study and contained an unprecedented wealth of yet uncharacterised transcripts. Guided by these data, I performed a GFP-tagging screen identifying two proteins which may play an important role in L. mexicana biology, LmxM.16.0500, a member of a small, divergent, amastin-derived gene family, which appears to be released from the cell body of PRO, and LmxM.09.1330 a specific marker of the amastigote flagellar pocket.
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Barão, Sandra Cristina. "Prevalencia da infecção por Leishmania chagasi em area de autoctonia recente, Araçatuba/SP". [s.n.], 2007. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311143.
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Orientador: Mariangela Ribeiro ResendeTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: As informações que existem acerca da leishmaniose visceral humana, provêm em sua maioria das notificações realizadas nas áreas de alta endemicidade. Por isso, ainda há muitos aspectos a respeito da transmissão urbana e dos quadros de infecção assintomática que precisam ser elucidados. O dimensionamento real da prevalência da infecção por Leishmania chagasi pode contribuir para a definição e avaliação do impacto das medidas de controle. Com o objetivo de determinar a prevalência da infecção por L. chagasi em área de autoctonia recente, município de Araçatuba e, avaliar os fatores associados em relação aos casos humanos de leishmaniose visceral notificados, foi realizado um estudo transversal, com amostra estratificada de fase única, realizada em duas áreas urbanas de níveis sócio-econômicos distintos, designadas A1 (periférica, menor nível sócio-econômico) e A2 (central, melhor nível sócio-econômico). A soroprevalência foi avaliada com a utilização do teste imunocromatográfico com antígeno recombinante K39 (Ag-RK39). A prevalência observada foi de 18,4% (23/125) em A1 e 4,8% (6/125) em A2. A proporção entre indivíduos assintomáticos e casos de doença ativa nas áreas 1 e 2 foram respectivamente 1,35:1 e 2:1. Não houve diferença significativa da soropositividade na distribuição por idade, nem por sexo, entre as áreas. Contudo, foi observada diferença na proporção de casos assintomáticos entre as áreas, possivelmente associada aos níveis sócio-econômicos e intensidade de transmissão. Também houve relação com a presença canina nos últimos dois anos e a soropositividade para o Ag-rK39. As informações obtidas sugerem a associação da soroprevalência à presença canina nos dois últimos anos e reforça a estratégia de controle adotada
Abstract: Many information exist about human visceral leishmaniasis are origin to thepontificated cases, moreover, almost all data substantiating derive high levels transmission. So, there are many aspects about the urban transmission and asymptomatic infection to need to elucidated. The real comprehensive measurements about the Leishmania chagasi infection to be able to contribute to improve the assessment impact about the measures control. The objective to this study was determining the prevalence of asymptomatic visceral leishmaniasis infection in Araçatuba city, a recent autoctone area. This was a cross-sectional survey on a random sample of the population in two urban different areas, called A1 (outskirts, low social-economic condition) and A2 (central, good social-economic condition). The seroprevalence was assessing by the Immunochromatographic test with recombinant antibody K39 dipstick. The prevalence was 18.4% (23/125) in A1 and 4.8% (6/125) in A2. And the proportion between the asymptomatic and active disease in areas 1 and 2 was 1.35:1 and 2:1, respectively. There was no significant difference in age, nor gender, distribution of seropositivity between the areas. But we observed a difference in asymptomatic infection rates between the two areas, possibly associated with socioeconomic levels and transmission intensity. The data from this study suggest an associate between the human symptomatic seroprevalence and the presence of dogs in last two years old
Doutorado
Ciencias Basicas
Doutor em Clínica Médica
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Silva, Ryan Emiliano da. "Genes de cisteíno proteases (catepsina L-like) de Leishmania infantum chagasi: caracterização, relações filogenéticas e diagnóstico molecular". Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-12072018-145115/.
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Os parasitas pertencentes ao gênero Leishmania têm distribuição ubíqua. Este táxon inclui Leishmania infantum chagasi, agente etiológico da leishmaniose visceral nas Américas, uma zoonose negligenciada cujas metodologias diagnósticas acumulam uma série de limitações, requerendo a validação e padronização de metodologias diagnósticas satisfatórias. Vários fatores estão relacionados à patogênese causada por este protozoário, entre eles a catepsina L-like, uma cisteíno protease envolvida em processos regulatórios metabólicos e infecciosos. Portanto, este trabalho teve como objetivo avaliar a eficácia do gene de catepsina L-like isoforma CPA como alvo de diagnóstico molecular e como marcador filogenético que permita a compreensão das variações intraespecíficas e elucidem a história evolutiva de L. infantum chagasi no Brasil. Foram utilizados 44 isolados de L. infantum chagasi de diferentes estados brasileiros. Os fragmentos do gene de catepsina L-like foram amplificados, purificados, sequenciados, alinhados manualmente e analisados por métodos filogenéticos de máxima parcimônia e inferência bayesiana. As sequências geradas foram usadas para pesquisar e sintetizar iniciadores a serem usados em reações específicas para o parasita alvo. O gene de catepsina L-like não mostrou variabilidade intraespecífica entre os isolados analisados, sugerindo um evento recente de introdução do mesmo nas Américas. O par de iniciadores propostos amplificou o DNA alvo de isolados de L. infantum chagasi, sendo efetivo na amplificação de DNA em concentrações de até 10-11g / µl. O marcador proposto não apresentou reações cruzadas com outros hemoparasitas de importância clínica. Quando utilizado para o diagnóstico em um painel de amostras clínicas de cães, obteve-se uma frequência de positividade de 49,03% (102/208), contrastando com o valor de 14,42% (30/208) obtido com o marcador para o gene do espaçador ribossomal interno ITS. Quando testado em amostras de flebotomíneos se obteve um valor de 6,25% e em amostras de pacientes humanos o valor foi de 14,28%. Os marcadores também foram eficazes em amplificar DNA extraído de amostras de urina, de sangue fixado em papel filtro e mesmo em amostras de swab de lesões conjuntivas. Este conjunto de parâmetros permite inferir que o CatLeish- PCR é sensível e específico para o diagnóstico de L. infantum chagasi podendo ser aplicado tanto em pesquisas clínicas quanto em inquéritos epidemiológicos de vigilância.The parasites belonging to the Leishmania genus have a wide distribution. This taxon includes Leishmania infantum chagasi, the etiologic agent of Visceral Leishmaniasis in the Americas, a neglected zoonosis that requires the validation and standardization of satisfactory diagnostic methodologies. Several factors are related to the pathogenesis caused by this protozoan, as Catepsin L-like, a cysteine protease involved in regulatory and infectious processes. Given this information this work aimed to evaluate the effectiveness of Cathepsin L-like isoform CPA as a target for molecular diagnosis and as a phylogenetic marker that allows understanding the intraspecific variations and the evolutionary history of L. infantum chagasi in Brazil. We used 44 isolates of L. infantum chagasi from different Brazilian states. The cathepsin L-like gene fragments were amplified, purified, sequenced, manually aligned and analyzed by maximum parsimony and Bayesian inference methods. The sequences generated were researched to construction of oligonucleotide primers to be used in reactions specific to the target parasite. The Cathepsin L-like gene did not show intraspecific variability among the isolates analyzed, suggesting a recent event of introduction of the same in the Americas. The pair of proposed primers amplified the target DNA of L. infantum chagasi isolates, being effective in DNA amplification at concentrations of up to 10-11g/µl. The proposed marker did not present cross-reactions with other hemoparasites of clinical importance. When used for the diagnosis in a panel of clinical samples of dogs obtained a positive frequency of 49.03% (102/208), against the 14.42% (30/208) to ribosomal ITS marker. Samples of sandflies obtained a value of 6.25% and in humans the value was 14.28%. The markers were also effective in blood samples fixed on filter paper and even in samples from conjunctival lesion swabs. This set of parameters allows to infer that CatLeish-PCR is a sensitive and specific marker for the diagnosis of L. infantum chagasi in clinical and epidemiological surveys.
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Pedron, Julien. "Synthèse et étude de l'activité anti-kinétoplastidés de nouvelles 8-nitroquinoléin-2(1H))-ones bioactivées par les nitroréductases de type 1". Thesis, Toulouse 3, 2018. http://www.theses.fr/2018TOU30190/document.
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Les kinétoplastidés sont des protozoaires flagellés responsables de maladies tropicales négligées mortelles telles que la leishmaniose viscérale (L. donovani et L. infantum) ou la trypanosomiase humaine africaine (T. brucei), pour lesquelles les traitements disponibles sont très limités. Depuis quelques années, on observe un regain d'intérêt pour le développement de nitrohétérocycles aromatiques anti-infectieux tels que le delamanide et le féxinidazole. De récentes études indiquent que l'activité anti-kinétoplastidés de ces dérivés repose sur leur bioactivation sélective par des nitroréductases parasitaires, conduisant à la formation de métabolites réduits électrophiles, fortement cytotoxiques. Suite à des études préliminaires réalisées dans notre équipe en série 8-nitroquinoléin-2(1H)-one, ces travaux de thèse portent sur la synthèse et l'étude in vitro de l'activité antiparasitaire de 80 dérivés notamment fonctionnalisés en positions 3 et 6 du pharmacophore par divers motifs, notamment via la mise au point de réactions d'halogénation sélective et de couplages pallado-catalysés. Ainsi, 5 nouvelles molécules hits (4 anti-kinétoplastidés et 1 sélective de T. brucei) ont été identifiées (0,01 µM ≤ CI50 ≤ 7 µM et 13 < IS < 1500), trois d'entre-elles étant des substrats sélectifs des nitroréductases parasitaires de type I. Afin de préciser les relations structure-activité, une étude des potentiels de réduction a également été menée. Des études physico-chimiques (solubilité, test de perméabilité PAMPA) et pharmacocinétiques in vitro (stabilité microsomale et fixation à l'albumine humaine) sont venues compléter ce travail. Enfin, des évaluations de la mutagénicité et de la génotoxicité de ces hits sur des cellules procaryotes et humaines ont été conduites, dans le but de statuer sur leur potentiel pharmaceutique antiparasitaire humain et vétérinaireKinetoplastids are flagellated protozoan parasites responsible for lethal neglected tropical diseases, such as visceral leishmaniasis (L. donovani and L. infantum) or sleeping sickness (T. brucei brucei), for which very few drugs are available. Nowadays, nitroheterocyclic compounds present a renewed interest as anti-infective agents, as illustrated by the development of fexinidazole and delamanid. Some recent studies demonstrated that the antikinetoplastid activity of these derivatives involves their selective bioactivation by parasitic nitroreductases, leading to the formation of electrophilic reduced metabolites, highly cytotoxic. Based on preliminary studies conducted in our team in 8-nitroquinolin-2(1H)-one series, this PhD work is about the synthesis and in vitro antiparasitic study of 80 derivatives mainly functionalized at positions 3 and 6 of the pharmacophore by various substituents, especially via the optimization of selective halogenation and pallado-catalyzed cross coupling reactions. Thereby, 5 new hit compounds (4 antikinetoplastid and 1 selective of T. brucei) were identified (0.01 µM ≤ IC50 ≤ 7 µM and 13 < SI < 1500), three of them being selective substrates of type I parasitic nitroreductases. In order to refine the structure-activity relationship studies, an analysis of reduction potentials was also conducted. In vitro physicochemical (solubility, PAMPA permeability assay) and pharmacokinetic (microsomal stability and human albumin binding) experiments completed this work. Finally, the mutagenicity and genotoxicity evaluations of these new hit compounds toward prokaryotic and human cells were realized, in order to assess their human and veterinary antiparasitic pharmaceutical potential
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MARITATI, MARTINA. "LEISHMANIASIS: A RE-EMERGING NEGLECTED DISEASE. BIOMOLECULAR AND METABOLOMIC STUDIES AIMED AT IMPLANTING ITS CONTROL IN EMILIA-ROMAGNA". Doctoral thesis, Università degli studi di Ferrara, 2021. http://hdl.handle.net/11392/2487987.
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Background. Leishmaniases (VL) represents a major health problem.
The first part of the thesis evaluates by molecular techniques and cytokine analysis the prevalence of asymptomatic Leishmania infections in autoimmune rheumatic patients treated with biological drugs and living in Leishmania endemic foci in Italy.
In the second part, the effect of the niacin analogue, 6-AN on Leishmania parasite growth and metabolism using the metabolomics technology was investigated. The pentose phosphate pathway (PPP), has been reported as a target of 6-AN, thus PPP might represent a good target.
Methods. VL qualitative and real-time PCR were performed on DNA extracted from PBMCs from 50 autoimmune rheumatic patients treated with immunosuppressive biologic drugs for at least 5 years. Plasma cytokine concentrations were also measured in plasma from Leishmania DNA-positive and -negative rheumatic patients as well as from the healthy control group.
In the 6-AN study, L. mexicana M379 and L. infantum PCM5 promastigotes were treated with 7.8 mM 6-AN and 2.17% DMSO for 24 hours. After vitality, infectivity of 6-AN-treated promastigotes to mouse macrophages, and 6-AN interactions with oxidizing compounds were also studied. Small metabolites were extracted and analysed by pHILIC-LC-MS in polarity switching mode and data were analysed with IDEOMv19 and MetaboAnalyst 3.0.
Results.Eighteen out the 50 (36%) autoimmune rheumatic patients were positive for Leishmania DNA by conventional and/or quantitative PCR with a detection of high parasite burdens (1 to 136 parasite/ml in 4 patients, 1.000 to 40.000 in 11 patients and over 1.000.000 in 3 patients). Patients that were taking a steroid in association with the biological drug showed a higher positivity for circulating L. infantum kDNA than those given the biological drug only (p<0.05). Pro-inflammatory IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ and TNF-α; anti-inflammatory IL-4, IL-13; and regulatory IL-10 cytokines were markedly elevated in all autoimmune rheumatic patients with additional increases in inflammatory mediators in autoimmune rheumatic patients positive for Leishmania DNA.
In both L. mexicana and L. infantum, 6-AN caused significant depletion of phosphoribosylpyrophosphate (PRPP) and nicotinate (Na) and as a result purine and pyrimidine nucleotides were reduced and their nucleobases accumulated. Glutathione, ribose-5-phosphate, 6-phosphogluconate levels and downstream PPP intermediates were similar to controls. For L. infantum, it was possible to analyse NAD+ and NADPH, which were found decreased together with the PPP intermediate D-sedoheptulose 7-phosphate. Moreover, 6-AN treatment caused a marked elongation in parasite body. 6-AN in combination with the oxidizing compounds has additive effects against Leishmania and did not affect the infectivity of the treated promastigotes to mouse macrophages.
Conclusions.VL molecular screening and cytokine analysis should be taken into account before treating autoimmune rheumatic patients with biologic drugs, especially in rural areas.
In mammals 6-AN is converted to abnormal 6-ANAD/P by NAD+ glycohydrolase, however, in Leishmania its toxicity is only seen in millimolar range, in which 6-AN is responsible for the depletion of cellular phosphoribosyl pyrophosphate (PRPP) content probably in the Preiss-Handler NAD+ salvage pathway, resulting in depletion of nucleotides required for nucleic acid biosynthesis. The marked elongation in the 6-AN-treated parasite bodies confirms nucleotide starvation. Leishmania NAD+ glycohydrolase might decompose NAD+ but might not catalyze exchange reactions, as found in other microrganisms, however, combined 13C-glucose labeling and flux analysis might be useful to ascertain the fate and action mechanism of 6-AN in Leishmania. In addition, PRPP synthetase should also be a good target for new potential drugs against leishmaniasis pointing to the growth-inhibitory effect of PRPP depletion.Background. Leishmaniases (VL) represents a major health problem. The first part of the thesis evaluates by molecular techniques and cytokine analysis the prevalence of asymptomatic Leishmania infections in autoimmune rheumatic patients treated with biological drugs and living in Leishmania endemic foci in Italy. In the second part, the effect of the niacin analogue, 6-AN on Leishmania parasite growth and metabolism using the metabolomics technology was investigated. The pentose phosphate pathway (PPP), has been reported as a target of 6-AN, thus PPP might represent a good target. Methods. VL qualitative and real-time PCR were performed on DNA extracted from PBMCs from 50 autoimmune rheumatic patients treated with immunosuppressive biologic drugs for at least 5 years. Plasma cytokine concentrations were also measured in plasma from Leishmania DNA-positive and -negative rheumatic patients as well as from the healthy control group. In the 6-AN study, L. mexicana M379 and L. infantum PCM5 promastigotes were treated with 7.8 mM 6-AN and 2.17% DMSO for 24 hours. After vitality, infectivity of 6-AN-treated promastigotes to mouse macrophages, and 6-AN interactions with oxidizing compounds were also studied. Small metabolites were extracted and analysed by pHILIC-LC-MS in polarity switching mode and data were analysed with IDEOMv19 and MetaboAnalyst 3.0. Results.Eighteen out the 50 (36%) autoimmune rheumatic patients were positive for Leishmania DNA by conventional and/or quantitative PCR with a detection of high parasite burdens (1 to 136 parasite/ml in 4 patients, 1.000 to 40.000 in 11 patients and over 1.000.000 in 3 patients). Patients that were taking a steroid in association with the biological drug showed a higher positivity for circulating L. infantum kDNA than those given the biological drug only (p<0.05). Pro-inflammatory IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ and TNF-α; anti-inflammatory IL-4, IL-13; and regulatory IL-10 cytokines were markedly elevated in all autoimmune rheumatic patients with additional increases in inflammatory mediators in autoimmune rheumatic patients positive for Leishmania DNA. In both L. mexicana and L. infantum, 6-AN caused significant depletion of phosphoribosylpyrophosphate (PRPP) and nicotinate (Na) and as a result purine and pyrimidine nucleotides were reduced and their nucleobases accumulated. Glutathione, ribose-5-phosphate, 6-phosphogluconate levels and downstream PPP intermediates were similar to controls. For L. infantum, it was possible to analyse NAD+ and NADPH, which were found decreased together with the PPP intermediate D-sedoheptulose 7-phosphate. Moreover, 6-AN treatment caused a marked elongation in parasite body. 6-AN in combination with the oxidizing compounds has additive effects against Leishmania and did not affect the infectivity of the treated promastigotes to mouse macrophages. Conclusions.VL molecular screening and cytokine analysis should be taken into account before treating autoimmune rheumatic patients with biologic drugs, especially in rural areas. In mammals 6-AN is converted to abnormal 6-ANAD/P by NAD+ glycohydrolase, however, in Leishmania its toxicity is only seen in millimolar range, in which 6-AN is responsible for the depletion of cellular phosphoribosyl pyrophosphate (PRPP) content probably in the Preiss-Handler NAD+ salvage pathway, resulting in depletion of nucleotides required for nucleic acid biosynthesis. The marked elongation in the 6-AN-treated parasite bodies confirms nucleotide starvation. Leishmania NAD+ glycohydrolase might decompose NAD+ but might not catalyze exchange reactions, as found in other microrganisms, however, combined 13C-glucose labeling and flux analysis might be useful to ascertain the fate and action mechanism of 6-AN in Leishmania. In addition, PRPP synthetase should also be a good target for new potential drugs against leishmaniasis pointing to the growth-inhibitory effect of PRPP depletion.
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Kanwar, Ankush. "Studies Aimed at the Synthesis of Anti-Infective Agents". Scholar Commons, 2018. http://scholarcommons.usf.edu/etd/7176.
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Infectious diseases continue to be a major concern worldwide. They are the second leading cause of death after heart disease. Factors such as an increasing global population, travel, urbanization, global climate change and evolution of pathogens have made infectious diseases more common. Infectious diseases, particularly neglected tropical diseases (NTDs) result in many deaths worldwide. Malaria and leishmaniasis are two common (NTDs) which affect low income countries around the globe. Low cost drugs with novel mechanism of action are required to tackle the growing resistances of parasites against current drugs used in the developing world, where most of the cases occur. The first part of this manuscript (chapters 1 - 3) describes the synthesis of novel analogs active against Leishmania donovani parasite which causes leishmaniasis. Leishmaniasis is a vector-borne complex group of diseases transmitted through the bite of an infected female sand-fly. Its clinical manifestations range from the less severe (cutaneous) to fatal (visceral) forms depending upon infecting species, immunity of host and the environment. Reports have suggested the role of Heat shock protein 90 (Hsp 90) in the differentiation of the Leishmania parasite from the promastigote stage to the pathogenic amastigote stage inside the host. A series of tetrahydro-indazole, tetrahydro-pyrazolo pyridine and radicicol hybrid compounds were prepared based on known Hsp 90 inhibitors, SNX2112 and NVP-AUY922. The synthetic approach allowed us to generate a diverse library of analogs which were used to probe the hydrophobic pocket of Hsp 90 active site. The most active compound, was found to be twice more active as the clinically used drug, Miltefosine, in an infected macrophage assay with an IC50 = 0.88 µM.
The second part of this manuscript (chapters 4 - 5) describes the synthesis of xanthurenic acid analogs as antimalarial drugs. Xanthurenic acid (XA) is a vital component for the gametogenesis of the Plasmodium inside the mosquito’s gut. Gametogenesis plays an important part in the continuation of the parasite’s life cycle. A series of xanthurenic acid analogs were synthesized with the aim of inducing premature exflagellation of the microgametes, thus blocking the key step required for the transmission of parasites from humans to the mosquito. A biotinylated xanthurenic acid analog and a clickable xanthurenic acid analog were also synthesized which will help us investigate the mechanism of action of xanthurenic acid in inducing gametogenesis in mosquito. In the preliminary screening efforts in an exflagellation assay, analog 4.40 showed promising activity and was more active in inducing exflagellation than xanthurenic acid. An exflagellation assay of other analogs is currently being pursued. Further investigations into the molecular target and mechanism of action are underway with the biotinylated xanthurenic acid analog.
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BONETTI, FRANCO C. "Acao da radiacao ionizante sobre a morfologia, fisiologia e crescimento da Leishmania amazonensis, com avaliacao de seu poder imunogenico em modelos experimentais". reponame:Repositório Institucional do IPEN, 2002. http://repositorio.ipen.br:8080/xmlui/handle/123456789/11025.
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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN/CNEN-SP