Discovery of new drugs candidates for visceral leishmaniasis treatment : from image-based assay development to in vitro compounds screening

Orientador: Lucio Holanda Gondim de Freitas JuniorTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As leishmanioses são doenças negligenciadas causadas por protozoários do gênero Leishmania, que provocam grande impacto na saúde pública de países em desenvolvimento. A quimioterapia disponível ainda é limitada e apresenta importantes limitações; portanto, é prioritária a pesquisa e descoberta de novos fármacos com potencial leishmanicida. Nesse contexto, os ensaios de alto conteúdo (HCS) vem sendo empregados eficientemente nas fases iniciais do desenvolvimento de fármacos, para identificação de moléculas candidatas com atividade anti-Leishmania. Apesar das vantagens, essa metodologia ainda apresenta baixas taxas de hits e de confirmação, quando comparada a modelos de outras doenças, e alta taxa de falhas quando os compostos são testados em modelos in vivo. Além disso, os protocolos de HCS vem sendo empregados para um número limitado de cepas de Leishmania, geralmente L. donovani, embora espécies/cepas possam diferir drasticamente em termos de infecção e suscetibilidade aos fármacos. Dessa forma, focado em protocolos de HCS previamente publicados, um ensaio baseado em imagem foi desenvolvido e otimizado, para a triagem de compostos contra amastigotas intracelulares de diferentes cepas/espécies viscerais de Leishmania. Para cada espécie/cepa, estabeleceu-se o modelo biológico, determinando-se as melhores condições de cultivo para célula hospedeira e parasita, infecção e período de exposição aos compostos, e padronizou-se o método de análise de imagem através do software comercial. Entre as cepas/espécies estabelecidas, houve diferenças no perfil de infecção, em termos de taxa de infecção, número de parasitas intracelulares e taxa de multiplicação celular; bem como na atividade de compostos referência: enquanto a anfotericina B apresentou atividade consistente contra as cepas, a miltefosina mostrou atividade dependente da cepa, com valores de EC50 entre 0,4 e 7,6 µM. O mesmo perfil de variação dependente da cepa ocorreu na dinâmica de atividade dos compostos referência, na triagem de bibliotecas e na atividade dos hits selecionados; evidenciando, assim, a importância da expansão do portfólio de ensaios in vitro para Leishmania. Através da metodologia estabelecida, a prospecção de candidatos a fármacos foi realizada, utilizando diferentes abordagens: triagem de bibliotecas de extratos naturais e compostos sintéticos, reposicionamento de fármacos e combinação terapêutica. Destacam-se os resultados obtidos através: (i) da biblioteca Hypha Mycodiverse, da qual foram identificados cinco compostos purificados com atividade anti-Leishmania promissora (EC50 ? 1,3 µM), (ii) das coleções de compostos/extratos de diferentes colaboradores, com classes terapêuticas e químicas inéditas ou pouco exploradas, tais como marinoquinolinas, chalconas e belzil-farnesil aminas, e candidatos potenciais (EC50 < 10 µM), representando moléculas e scaffolds iniciais para otimização química, (iii) da biblioteca comercial LOPAC, da qual o composto nitro CB1954 apresentou atividade de amplo espectro: contra as diferentes cepas e formas do ciclo de vida do parasita e contra outros tripanossomatídeos, com valores de EC50 na grandeza de nano e micro molar, e (iv) da combinação terapêutica, na qual o inibidor de calpaína MDL28170 apresentou perfil sinérgico em associação com anfotericina e miltefosina. Conclui-se que a metodologia estabelecida representa uma importante contribuição para os processos iniciais do desenvolvimento de fármacos, sendo que os candidatos identificados nesse trabalho poderão ser explorados futuramente como alternativa de terapia para leishmaniosesAbstract: Leishmaniasis are neglected diseases caused by protozoan parasites of genus Leishmania, which lead to severe impact in public health of developing countries. The available chemotherapy is still inadequate and presents important limitations; therefore, the research and the discovery of new leishmanicidal drugs must be prioritized. In this context, the high content screenings (HCS) have been applied efficiently in the early drug development, for identification of candidate molecules with anti-Leishmania activity. Despite the advances, the methodology still has several drawbacks such as low ratio of hits and confirmation, compared to others diseases models, and high number of failures, when compounds are assessed in in vivo assays. Additionally, HCS protocols have been developed, to the date, for a limited number of Leishmania species, mostly L. donovani, although different species/strains may diverge dramatically in regard to drug susceptibility. Therefore, focused on HCS protocols previously published, an image-based assay was developed and optimized, in order to screen compounds against intracellular amastigotes of distinct Leishmania species and strains. For each species/strains, the biological model was established by determining the best conditions of host cell and parasite culture, infection and period of drug exposition; and an image analysis method was standardized using a commercial software. Among the strains/species established, differences were observed in the general infection parameters, in terms of infection ratio, intracellular amastigotes number and multiplying amastigotes ratio; also, diverging results were verified for reference compounds activity: while amphotericin B presented consistent activity against strains, miltefosine showed strain-dependent activity, with EC50 values from 0.4 to 7.6 µM. The strain-specific profile also occurred in the activity dynamics of reference compounds, in libraries screening and in the activity of selected hits, confirming the importance of expanding the in vitro Leishmania portfolio. Throughout this methodology, candidates¿ prospections were performed within different screening approaches: triage of libraries containing natural products and synthetic compounds, drugs repurposing and drugs combination. Among the results, we highlight: (i) Hypha Mycodiverse library, from which we identified five purified compounds with promising anti-Leishmania activity (EC50 ? 1.3 µM), (ii) Compounds and extracts collections from collaborators, from which we discovered new chemical and therapeutic classes, such as marinoquinolines, chalcones and belzil-farnesil amines, and potential candidates (EC50 < 10 µM), representing molecules and scaffolds for chemical optimization, (iii) LOPAC library screening, from which we selected the nitro compound CB1954, that presented a broad-spectrum activity: against distinct strains and life cycle forms of Leishmania and others trypanosomatids, with EC50 values from nano to micro molar and (iv) drugs combination, in which the calpain inhibitor MDL28170 present synergic effect in association with amphotericin B and miltefosine. We concluded that this novel methodological approach represents an important contribution for early stage anti-Leishmania drug discovery and the candidates identified can be further explored as an alternative for leishmaniasis therapyDoutoradoImunologiaDoutora em Genética e Biologia Molecular140907/2013-0CNP

Spontaneous cytokine production in children according to biological characteristics and environmental exposures.

BACKGROUND: Environmental factors are likely to have profound effects on the development of host immune responses, with serious implications for infectious diseases and inflammatory disorders such as asthma. OBJECTIVE: This study was designed to investigate the effects of environmental exposures on the cytokine profile of children. METHODS: The study involved measurement of T helper (Th) 1 (interferon-gamma), 2 [interleukin (IL)-5 and IL-13], and the regulatory cytokine IL-10 in unstimulated peripheral blood leukocytes from 1,376 children 4-11 years of age living in a poor urban area of the tropics. We also assessed the impact of environmental exposures in addition to biological characteristics recorded at the time of blood collection and earlier in childhood (0-3 years before blood collection). RESULTS: The proportion of children producing IL-10 was greater among those without access to drinking water [p < 0.05, chi-square test, odds ratio (OR) = 1.67]. The proportion of children producing IL-5 and IL-10 (OR = 10.76) was significantly greater in households that had never had a sewage system (p < 0.05, trend test). CONCLUSIONS: These data provide evidence for the profound effects of environmental exposures in early life as well as immune homeostasis in later childhood. Decreased hygiene (lack of access to clean drinking water and sanitation) in the first 3 years of life is associated with higher spontaneous IL-10 production up to 8 years later in life

Systematic Y2H screening reveals extensive effector-complex formation

During infection pathogens secrete small molecules, termed effectors, to manipulate and control the interaction with their specific hosts. Both the pathogen and the plant are under high selective pressure to rapidly adapt and co-evolve in what is usually referred to as molecular arms race. Components of the host’s immune system form a network that processes information about molecules with a foreign origin and damage-associated signals, integrating them with developmental and abiotic cues to adapt the plant’s responses. Both in the case of nucleotide-binding leucine-rich repeat receptors and leucine-rich repeat receptor kinases interaction networks have been extensively characterized. However, little is known on whether pathogenic effectors form complexes to overcome plant immunity and promote disease. Ustilago maydis, a biotrophic fungal pathogen that infects maize plants, produces effectors that target hubs in the immune network of the host cell. Here we assess the capability of U. maydis effector candidates to interact with each other, which may play a crucial role during the infection process. Using a systematic yeast-two-hybrid approach and based on a preliminary pooled screen, we selected 63 putative effectors for one-on-one matings with a library of nearly 300 effector candidates. We found that 126 of these effector candidates interacted either with themselves or other predicted effectors. Although the functional relevance of the observed interactions remains elusive, we propose that the observed abundance in complex formation between effectors adds an additional level of complexity to effector research and should be taken into consideration when studying effector evolution and function. Based on this fundamental finding, we suggest various scenarios which could evolutionarily drive the formation and stabilization of an effector interactome

RELATO DE CASO: Prática clínica de uso da Imunoglobulina Humana em paciente com neuropatia grave e perda motora severa após tratamento quimioterápico com Brentuximabe para Linfoma de Hodgkin/CASE REPORT: Clinical practice of using Human Immunoglobulin in a patient with severe neuropathy and severe motor loss after chemotherapy with Brentuximab for Hodgkin's Lymphoma

Segue relato de caso de um adolescente J.V.R.T.S. 16 anos, que após receber tratamento quimioterápico para Linfoma Hodgkin com Brentuximabe vedotina evoluiu com disestesias e tetraparesia flácida sugestivas de forma atípica da Síndrome de Guillain-Barré sendo tratado com Imunoglobulina Humana obtendo resolução completa do quadro.O trabalho foi realizado com o intuito de buscar embasamento científico para esclarecer a relação do Linfoma de Hodgkin, assim como do tratamento antineoplásico com a etiologia da neuropatia apresentada pelo paciente, explicar o mecanismo de ação desse tratamento nas neuropatias graves secundárias ao linfoma e seu tratamento focando na resolução rápida e completa desse caso, definir apresentação clinica das neuropatias secundárias e apresentação desta no paciente em questão diferenciando do padrão típico de guillain barré e definir quais causas de todo processo é mais importante: a própria relação do linfoma como síndrome paraneoplásica ou tratamento antineoplásico como gerador da neuropatia apresentada

Identification of Inhibitors to trypanosoma cruzi sirtuins based on compounds developed to human enzymes

Chagas disease is an illness caused by the protozoan parasite Trypanosoma cruzi, affecting more than 7 million people in the world. Benznidazole and nifurtimox are the only drugs available for treatment and in addition to causing several side effects, are only satisfactory in the acute phase of the disease. Sirtuins are NAD+-dependent deacetylases involved in several biological processes, which have become drug target candidates in various disease settings. T. cruzi presents two sirtuins, one cytosolic (TcSir2rp1) and the latter mitochondrial (TcSir2rp3). Here, we characterized the effects of human sirtuin inhibitors against T. cruzi sirtuins as an initial approach to develop specific parasite inhibitors. We found that, of 33 compounds tested, two inhibited TcSir2rp1 (15 and 17), while other five inhibited TcSir2rp3 (8, 12, 13, 30, and 32), indicating that specific inhibitors can be devised for each one of the enzymes. Furthermore, all inhibiting compounds prevented parasite proliferation in cultured mammalian cells. When combining the most effective inhibitors with benznidazole at least two compounds, 17 and 32, demonstrated synergistic effects. Altogether, these results support the importance of exploring T. cruzi sirtuins as drug targets and provide key elements to develop specific inhibitors for these enzymes as potential targets for Chagas disease treatment

CONSTRUCCIÓN Y VALIDACIÓN DE APLICACIÓN DIGITAL PARA ENSEÑANZA DE INSTRUMENTACIÓN QUIIRÚRGICA

Objetivo: construir y validar aplicación asociada a la enseñanza de instrumentación quirúrgica básica para académicos de enfermería.Método: estudio metodológico, desarrollado entre mayo y septiembre de 2016, en universidad pública de Piauí, Brasil. Se realizaron las etapas de análisis, proyecto, desarrollo y evaluación, con base en el referencial de Galvis-Panqueva. La validación de contenido y apariencia se hizo con 22 profesionales, y la evaluación con el público objetivo correspondió a 60 académicos de enfermería.Resultados: el nombre de la aplicación es “Instrumentais Cirúrgicos” y ella está en la plataforma Google play, disponible para uso en el sistema operacional Android. El Índice de Validad de Contenido global fue de 0,9 cuanto al contenido y 0,8 cuanto a la apariencia. El público objetivo evaluó la aplicación como excelente, cuanto a los criterios motivación, estilo y contenido, con promedio de 76,3%.Conclusión: la aplicación fue eficaz para utilización como herramienta auxiliar de enseñanza acerca del asunto instrumentación quirúrgica.Objetivo: construir e validar aplicativo sobre ensino de instrumentação cirúrgica básica para acadêmicos de enfermagem.Método: estudo metodológico, desenvolvido entre maio e setembro de 2016, em universidade pública do Piauí, Brasil. Seguiram-se as etapas de análise, desenho, desenvolvimento e avaliação, com base no referencial de Galvis-Panqueva. A validação de conteúdo e aparência foi realizada com 22 profissionais, e a avaliação com o público-alvo correspondeu a 60 acadêmicos de enfermagem.Resultados: o aplicativo foi nomeado de Instrumentais Cirúrgicos e está hospedado na plataforma Google play, disponível para uso no sistema operacional Android. Apresentou Índice de Validade de Conteúdo global de 0,9 quanto ao conteúdo e 0,8 quanto à aparência. O público-alvo avaliou o aplicativo como excelente, quanto aos itens de motivação, estilo e conteúdo, com média de 76,3%.Conclusão: o aplicativo mostrou-se válido para utilização como ferramenta auxiliar de ensino sobre o assunto instrumentação cirúrgica.Objective: To build and validate an application on teaching of basic surgical instrumentation to nursing students.Method: Methodological study conducted between May and September of 2016, at a public university in Piauí, Brazil. Analysis, design, development and evaluation stages were conducted based on the Galvis-Panqueva benchmark. Content and appearance validation was performed by 22 judges, and 60 nursing students selected by convenience sampling were the target audience that evaluated the instrument.Results: The application was named Surgical Instruments and is hosted on the Googleplay platform, available for use on the Android operating system. It had a global Content Validity Index of 0.9 for content and 0.8 for appearance. The target audience rated the application as excellent for motivation, style and content items, averaging 76.3%.Conclusion: The application has proved to be valid for use as an auxiliary teaching tool on the subject surgical instrumentation

Milk adulteration with acidified rennet whey: a limitation for caseinomacropeptide detection by high-performance liquid chromatography

peer-reviewedBACKGROUND High‐performance liquid chromatography (HPLC) is widely employed to determine the caseinomacropeptide (CMP) index and to detect milk tampering with rennet whey. Prior to HPLC analysis, CMP is subject to a trichloracetic acid isolation, causing further soluble proteins in the sample to precipitate. On this basis, we aimed to determine whether rennet whey acidification could adversely affect the HPLC sensitivity with respect to detecting this peptide. RESULTS As hypothesized, the CMP index from milk with added acidified rennet whey was, on average, half that quantified from milk with added rennet whey. Moreover, the quantum satis of acidified whey added to milk sufficient to demonstrate a HPLC CMP > 30 mg L–1 was 94% greater than that required for this threshold to be reached with rennet whey. CONCLUSION Milk tampering with acidified rennet whey may limit the analytical sensitivity of the reversed‐phase HPLC employed for the screening of CMP and, ultimately, disguise the fraudulent addition of whey to milk. © 2017 Society of Chemical IndustryCoordenação de Aperfeiçoamento de Pessoal de Nível Superio

Biomodelos Ósseos Produzidos por Intermédio da Impressão 3D: Uma Alternativa Metodológica no Ensino da Anatomia Veterinária

Recursos tecnológicos podem contribuir para o ensino da Anatomia Veterinária, tornando as disciplinas relativas a tal área, que são essenciais para a formação dos estudantes de Medicina Veterinária, cada vez mais atualizadas frente às novas tecnologias e às novas gerações de estudantes. Neste trabalho, o objetivo foi aplicar a digitalização e a impressão 3D para produzir biomodelos dos esqueletos canino e equino, de modo a disponibilizar as peças produzidas como ferramenta alternativa de estudo nas aulas práticas de anatomia veterinária. Ossos de cão e equino foram digitalizados, sendo possível realizar a sua impressão 3D com preservação eficaz das principais estruturas anatômicas. Por meio desse processo, também foi possível gerar arquivos digitais para que sejam utilizados durante as aulas práticas. Além disso, os biomodelos e os arquivos digitais produzidos poderão ser aplicados como uma forma alternativa e complementar para o estudo anatômico do esqueleto canino e equino.Technological resources can contribute to the teaching of Veterinary Anatomy, making this discipline, which is basic and essential for students of Veterinary Medicine, more interesting and accurate. In this study, the objective was to apply the 3D scanning and printing to produce biomodels of the canine and equine skeleton proposing to make available the models produced as a study tool in the practical classes of veterinary anatomy. Dog and equine bones were scanned. Then, it was possible to make the 3D printing of these bones with effective preservation of the main anatomical structures. Through scanning, it was also possible to generate digital files aiming their use in the classroom. These biomodels can be applied in the anatomical canine and equine skeleton study