Leishmaniasis is caused by protozoan parasites of the genus In mammalians, these parasites survive and replicate in macrophages and parasite elimination by macrophages is critical for host resistance. Endosomal Toll-like receptors (TLRs) have been shown to be crucial for resistance to For example, mice in the resistant C57BL/6 genetic background that are triple-deficient for TLR3, -7, and -9 () are highly susceptible to infection. mice are as susceptible as mice deficient in MyD88 or UNC93B1, a chaperone required for appropriate localization of endosomal TLRs, but the mechanisms are unknown. Here we found that macrophages infected with undergo autophagy, which effectively accounted for restriction of parasite replication. Signaling via endosomal TLRs was required for autophagy because macrophages deficient for TLR3, -7, and 9, UNC93B1, or MyD88 failed to undergo-induced autophagy. We also confirmed that ,, and cells were highly permissive to replication. Accordingly, shRNA-mediated suppression of Atg5, an E3 ubiquitin ligase essential for autophagosome elongation, in macrophages impaired the restriction of replication in C57BL/6, but did not affect parasite replication in or macrophages. Rapamycin treatment reduced inflammatory lesions formed in the ears of-infected C57BL/6 and mice, indicating that autophagy operates downstream of TLR signaling and is relevant for disease development Collectively, our results indicate that autophagy contributes to macrophage resistance to replication, and mechanistically explain the previously described endosomal TLR-mediated resistance to infection.
BackgroundThe interleukin 32 (IL-32) is a proinflammatory cytokine produced by immune and non-immune cells. It can be induced during bacterial and viral infections, but its production was never investigated in protozoan infections. American Tegumentary Leishmaniasis (ATL) is caused by Leishmania protozoan leading to cutaneous, nasal or oral lesions. The aim of this study was to evaluate the expression of IL-32 in cutaneous and mucosal lesions as well as in peripheral blood mononuclear cells (PBMC) exposed to Leishmania (Viannia) braziliensis.MethodsIL-32, tumour necrosis factor (TNF) and IL-10 protein expression was evaluated by immunohistochemistry in cutaneous, mucosal lesions and compared to healthy specimens. The isoforms of IL-32α, β, δ, γ mRNA, TNF mRNA and IL-10 mRNA were assessed by qPCR in tissue biopsies of lesions and healthy skin and mucosa. In addition, PBMC from healthy donors were cultured with amastigotes of L. (V.) braziliensis. In lesions, the parasite subgenus was identified by PCR-RFLP.ResultsWe showed that the mRNA expression of IL-32, in particular IL-32γ was similarly up-regulated in lesions of cutaneous (CL) or mucosal (ML) leishmaniasis patients. IL-32 protein was produced by epithelial, endothelial, mononuclear cells and giant cells. The IL-32 protein expression was associated with TNF in ML but not in CL. IL-32 was not associated with IL-10 in both CL and ML. Expression of TNF mRNA was higher in ML than in CL lesions, however levels of IL-10 mRNA were similar in both clinical forms. In all lesions in which the parasite was detected, L. (Viannia) subgenus was identified. Interestingly, L. (V.) braziliensis induced only IL-32γ mRNA expression in PBMC from healthy individuals.ConclusionsThese data suggest that IL-32 plays a major role in the inflammatory process caused by L. (Viannia) sp or that IL-32 is crucial for controlling the L. (Viannia) sp infection.
Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.
The multivalent effect of carbohydrates (glycoclusters) has been explored to study important biological targets and processes involving Trypanosoma cruzi (T. cruzi) infection. Likewise, CuAAC cycloaddition reactions (click chemistry) have been applied as useful strategy in the discovery of bioactive molecules. Hence, we describe the synthesis of 1,2,3-triazole-based tetravalent homoglycoclusters (1–3) and heteroglycoclusters (4 and 5) of d-galactopyranose (C-1 and C-6 positions) and sialic acid (C-2 position) to assess their potential to inhibit T. cruzi cell invasion and also its cell surface trans-sialidase (TcTS). The target compounds were synthesised in good yields (52–75 %) via click chemistry by coupling azidosugars galactopyranose and sialic acid with alkynylated pentaerythritol or tris(hydroxymethyl)-aminomethane (TRIS) scaffolds. T. cruzi cell invasion inhibition assays showed expressive low parasite infection index values (5.3–6.8) for most compounds. However, most glycoclusters proved to be weak TcTS inhibitors at 1 mM (<17 %), except the tetravalent sialic acid 3 (99 % at 1 mM, IC50 450 μM). Therefore, we assume that T. cruzi cell invasion blockage is not due to TcTS inhibition by itself, but rather by other mechanisms involved in this process. In addition, all glycoclusters were not cytotoxic and had significant trypanocidal activity upon parasite survival of amastigote forms.
Ao professor Dr. Marcelo Dias Baruffi pela orientação, pela oportunidade de desenvolver esse projeto e pela confiança depositada em mim e em meu trabalho. Agradeço pelo aprendizado e pelo exemplo de dedicação e amor à pesquisa. Ao Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e à Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pelo auxílio financeiro com a bolsa de doutoradoo. Ao Henrique e à Ana Lúcia, secretários do Programa de Pós-graduação em Biociências e Biotecnologia, por sempre se mostrarem prestativos em ajudar e esclarecendo minhas dúvidas. Aos professores Dr. Sérgio de Albuqurque, Dr. Emerson Bernardes Drª Leandra Ramalho e Drª Lúcia Faccioli pela colaboração no desenvolvimento deste trabalho e constante disponibilidade em ajudar. Um agradecimento especial à professora Leandra pelo axílio nas análises histopatológicas das lâminas de histologia e por sempre esclarecer as minhas dúvidas. À Vani Maria Alves Côrrea, funcionária do Departamento de Biologia Celular da FMRP-USP, pelo auxílio na montagem e coloração das lâminas de histologia. À Fabiana Rossetto Morais pelo auxílio técnico na citometria de fluxo e pelos momentos de descontração. Á Dona Regina, funcionária da limpeza, e ao Reinaldo, Ronaldo e Fábio, funcionários do Biotério, os quais possibilitaram a boa qualidade desse trabalho. Em especial aos funcionários do biotério pelo auxílio na manutenção dos camundongos utilizados neste trabalho. Ao Rubens Eduardo e à Lílian Cataldi pelo apoio técnico no laboratório. Aos meus amigos do Laboratório de Glicobiologia: Fábio, Luisa, Martin, Talícia, Tálita, Thais Canassa, Thais Plepis e Thalita pelas contribuições científicas, pelas discussões sobre experimentos, pela ajuda, pelo companheirismo, pelas risadas e pela amizade de todos. Agradeço especialmente à Thalita pela amizade e pela constante disponibilidade em ajudar. A todos os colegas do doutorado, em especial à Fabiana Zambuzi pela amizade, apoio e momentos agradáveis proporcionados. Aos meus queridos pais, Aparecida e Divino (in memorian), pelo amor incondicional e por me proverem a melhor educação possível, mesmo nos momentos mais FLEURI, A. K. A. Agradecimentos ix difíceis, nunca falhando. Serei eternamente grata, espero um dia poder retribuir todo o bem que vocês me deram. Eu amo vocês! Ao meu tio Marcelo e à minha avó por sempre me apoiarem nas minhas decisões e pelo apoio para eu continuar. Agradeço especialmente ao meu tio Marcelo, meu amigo e segundo pai, pela presença mesmo estando distante e pelo apoio de sempre em tudo. Aos meus irmãos, Jaqueline e Mateus, por me aturarem nos momentos difíceis e pelos ótimos momentos juntos, mesmo à distância. Agradeço especialmente à Jaqueline, minha irmã, amiga e cúmplice, por sempre me fortalecer na realização dos meus sonhos. Amo-te demais e para sempre! Ao meu noivo, Rafael, por me aturar de forma heroica em todos os momentos, por sempre estar disposto a me ajudar em qualquer situação e por acreditar em mim. Você me faz sentir a pessoa mais especial desse mun...
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