Atividade antimicobacteriana in vitro de lactonas sesquiterpênicas e investigação do seu mecanismo de ação

Tese (doutorado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2016.Introdução: A Tuberculose (TB) é uma doença infectocontagiosa considerada um problema global de saúde pública. A terapia em longo prazo, somada aos efeitos adversos dos medicamentos, contribui para a não adesão do paciente, o que resulta em falência do tratamento e seleção de linhagens de Mycobacterium tuberculosis resistentes. Por isso, a pesquisa e desenvolvimento de novos fármacos, que permitam a redução do tempo de tratamento e sejam uma alternativa para o tratamento de cepas resistentes, são considerados urgentes. Produtos naturais representam importantes fontes para o desenvolvimento de novos fármacos. A atividade antimicobacteriana já foi relatada para diversos compostos pertencentes a diferentes classes químicas, isolados de plantas, fungos e organismos marinhos, o que estimula a pesquisa de novos fármacos para o tratamento da TB provenientes de fontes naturais. Objetivos: Avaliar atividade antimicobacteriana in vitro de extratos e compostos naturais isolados de Calea uniflora e Calea pinnatifida e investigar seu mecanismo de ação. Métodos: Inicialmente, foi realizada uma triagem de 10 extratos e 11 compostos isolados obtidos das plantas Calea uniflora e Calea pinnatifida quanto à atividade antimicobacteriana in vitro frente a cepas referência de M. tuberculosis, Mycobacterium avium e Mycobacterium fortuitum, em concentrações entre 1,56 µg/mL e 100 µg/mL. Sete lactonas sesquiterpênicas (LS) apresentaram os melhores resultados e foram escolhidas para seguimento dos estudos da atividade antimicobacteriana in vitro, além da realização de análises in silico e estudo do seu mecanismo de ação, por meio da avaliação do perfil transcricional de M. tuberculosis após o tratamento com os compostos. Resultados: As LS (LS01 a LS07) apresentaram atividade antimicobacteriana frente a M. tuberculosis em fase exponencial de crescimento, de modo tempo e dose dependentes, com concentração inibitória mínima (MIC) variando de 6,25 µg/mL a 25 µg/mL. LS06 e LS07 também apresentaram atividade frente a M. tuberculosis em estado não-replicativo, em concentrações correspondentes a 4xMIC e 8xMIC, respectivamente. Nenhum dos compostos apresentou atividade frente a M. avium e M. fortuitum. Quando testados frente a isolados clínicos resistentes aos fármacos, os compostos apresentaram MIC semelhante às obtidas com os isolados sensíveis. Quando em combinação com isoniazida (INH) e rifampicina (RMP), os compostos não apresentaram sinergismo nem antagonismo. Os índices de seletividade variaram entre 1,77 a 11,88 em células de linhagem pulmonar MRC5. LS01, L06 e LS07 também apresentaram atividade frente a bacilos intracelulares, em modelo de infecção de macrófagos e demonstraram capacidade de proteção às células infectadas em concentrações correspondentes à MIC. Já quando em concentrações superiores, apresentaram citotoxicidade. Quando testada em estrutura granuloma-like, LS01 foi capaz de reduzir a contagem de unidades formadoras de colônia (UFC) na concentração 100 µM. As predições in silico indicaram que as LS estudadas neste trabalho podem apresentam boa biodisponibilidade por via oral, entretanto, podem sofrer intenso metabolismo de primeira passagem e apresentar altas taxas de ligação a proteínas plasmáticas. A análise do perfil transcricional de M. tuberculosis após o tratamento com LS01, LS06 e LS07 sugere que sua resposta pode estar relacionada à geração de espécies reativas de oxigênio (EROs), o que foi confirmado posteriormente, pela detecção de H2O2 em culturas de M. tuberculosis tratadas com os compostos. Este perfil também pode estar associado à habilidade das LS de se ligar aos resíduos de cisteína presentes em proteínas e no micotiol. Conclusão: Os compostos LS01-LS07 apresentaram boa atividade antimicobacteriana in vitro e parecem ter um novo mecanismo de ação quando comparadas aos fármacos utilizados atualmente no tratamento da TB e àqueles que se encontram em estudo clínico.Abstract : Background: Tuberculosis (TB) is a serious public health problem worldwide with one-third of the world´s population estimated to be infected with Mycobacterium tuberculosis. TB treatment requires the use of multiple drugs for at least 6 months. This lengthy drug therapy and adverse reactions to the drugs contribute to patient non-compliance, resulting in treatment failure and emergence of M. tuberculosis drug-resistant strains. There is an urgent need for new drugs to treat TB and natural products represent an important source for drug discovery. Several chemical classes of compounds isolated from plants, fungi and marine organisms have been described as potential antitubercular drugs, stimulating the search for new antimycobacterial agents isolated from natural sources. Aims: To evaluate the in vitro antimycobacterial activity of plant extracts and isolated plant-derived compounds from Calea uniflora and Calea pinnatifida and investigate their mode of action. Methods: A primary screening was conducted with 10 extracts from C. uniflora and C. pinnatifida and 12 purified compounds against M. tuberculosis, Mycobacterium avium and Mycobacterium fortuitum at concentrations ranging from 1.56 to 100 µg/mL. Seven sesquiterpene lactones (LS) showed the best results and were chosen for following studies on the in vitro antimycobacterial activity and also for in silico tests and transcriptional profiling analysis, in order to investigate their mode of action. Results: The LS (LS01-LS07) showed antimicrobial activity against log phase M. tuberculosis in a time- and concentration- dependent manner, with minimum inhibitory concentration (MIC) ranging from 6.25 to 25 µg/mL. LS06 and LS07 were also active against non-replicating M. tuberculosis at 4xMIC and 8xMIC, respectively. None of the compounds were active against M. avium and M. fortuitum. When tested against MDR M. tuberculosis clinical isolates, the compounds showed similar MIC compared to the sensitive isolates. They did not show synergism nor antagonism in combination with isoniazid or rifampicin. The selectivity index ranged from 1.77 to 11.88 in MRC5 lung cell line.LS01, LS06 and LS07 were also active against intracellular M. tuberculosis and were able to protect the infected cells at their MIC. Higher concentrations were found to be cytotoxic. When tested in a granuloma-like structure, LS01 reduced de colony forming units (CFU) counting at 100 µM. In silico studies indicate that these compounds may have good oral bioavailability, but intense first pass metabolism and high rates of plasma proteins binding. The transcriptional profiling analysis showed the response to LS01, LS06 and LS07 may be related to reactive oxygen species (ROS) generation, which was confirmed by the detection of H2O2 in M. tuberculosis cultures treated with the compounds. This response also may be related to the ability of the LS to bind to cysteine residues present in proteins and mycothiol. Conclusion: The LS 01-07 showed good in vitro antimycobacterial activity and seem to have a novel mode of action compared to the drugs currently used for the TB treatment and compared to the TB pipeline, which made them promising molecules for further studies

Isolamento e identificação de micobactérias não-tuberculosas em laboratório e hospital de referência do Estado de Santa Catarina

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em FarmáciaIntrodução: O gênero Mycobacterium compreende mais de 150 espécies, incluindo patogênicas, oportunistas e não patogênicas. As micobactérias pertencentes ao Complexo M. tuberculosis (CMTB) são responsáveis por causar tuberculose (TB), enquanto as Micobactérias Não-Tuberculosas (MNT) podem causar micobacterioses. Embora sejam clinicamente indistinguíveis, micobacterioses e TB diferem em relação ao tratamento. O diagnóstico convencional das micobactérias apresenta limitações quanto ao tempo de execução e à operacionalidade, visto que o resultado pode levar até 60 dias para ser liberado. Entretanto, o diagnóstico rápido e a identificação da espécie são fundamentais para a adoção da terapia adequada e consequente controle da doença. Objetivos: realizar a identificação de MNT e determinar a variabilidade das espécies isoladas no Laboratório de Referência do Estado de Santa Catarina, verificar a ocorrência de MNT em pacientes com diagnóstico de TB internados no Hospital de Referência para Tuberculose do Estado de Santa Catarina e avaliar o rendimento de técnicas moleculares para diagnóstico e identificação de micobactérias. Métodos: Oitenta e oito isolados de MNT, obtidos junto ao Laboratório de Referência do Estado de Santa Catarina, entre agosto de 2009 e agosto de 2011, foram identificados utilizando-se os métodos MMSA e PRA-hsp65. Amostras de escarro foram coletadas de 57 pacientes com diagnóstico de TB, internados no Hospital de Referência do Estado de Santa Catarina entre abril de 2010 e abril de 2011. A identificação das espécies foi realizada pelos métodos MMSA e PRA-hsp65. Estas amostras também foram utilizadas para a avaliação do rendimento das técnicas de PCR convencional, PCR em tempo real, PRA-hsp65 e MMSA, diretamente da amostra clínica. Resultados: Dentre os isolados de MNT recebidos do Laboratório de Referência, M. avium foi a espécie de maior ocorrência (39,8% dos casos), seguida de M. fortuitum (14,8%), M. abscessus (12,5%) e M. kansasii (6,8%). O pulmão foi o principal sítio de isolamento (85,3% dos casos) e 61,4% das amostras eram procedentes das regiões da Grande Florianópolis e do Vale do Itajaí. Do total de isolados de MNT, 34,2% foram associados ao desenvolvimento de doença. O Complexo M. avium foi o principal agente identificado (65,4% dos casos) e o pulmão foi o sítio de infecção mais frequente (88,6% dos casos). Houve predomínio do gênero masculino e de pacientes HIV-soronegativos e a média de idade foi de 46,1 ± 14,6 anos. Dos pacientes internados com diagnóstico de TB no Hospital de Referência, em apenas um foi identificada a presença de MNT. As técnicas de PCR e PCR em tempo real, utilizadas para detecção de micobactérias diretamente de amostras de escarro, apresentaram sensibilidade de 91,2% e 100%, respectivamente, tomando-se como padrão áureo o cultivo em meio sólido. Os métodos MMSA e PRA-hsp65 identificaram a espécie de micobactéria, diretamente de amostras de escarro, em 50,9% e 47,4% dos casos, respectivamente. Quando associados, possibilitaram a identificação em 75,4% das amostras. Conclusão: O presente estudo permitiu uma avaliação da realidade epidemiológica das MNT no Estado de Santa Catarina, que até então não havia sido estudada. Além disso, permitiu a avaliação de técnicas moleculares que podem ser utilizadas como ferramentas para a agilização do diagnóstico e da identificação de micobactérias.Background: The genus Mycobacterium comprises more than 150 species. The Mycobacterium tuberculosis Complex (MTC) causes tuberculosis (TB), while the Nontuberculous Mycobacteria (NTM) can cause mycobacteriosis. These diseases are clinically indistinguishable, but require different therapeutic regimens. The conventional diagnosis of mycobacteria is time-consuming and may take up 60 days. However, early diagnosis and identification followed by appropriate treatment are essential for an effective control of these diseases. Objectives: to identify and study the variability of NTM species isolated at the Reference Laboratory of Santa Catarina State, to find the occurrence of NTM among patients hospitalized with TB diagnosis in the Reference Hospital of Santa Catarina State and to estimate the yield of molecular techniques to early diagnosis and identification of mycobacteria. Methods: A total of 88 NTM isolates, obtained from Reference Laboratory between August 2009 and August 2011, were identified using MMSA and PRA-hsp65 methods. Sputum samples were collected from 57 patients with TB diagnosis who were hospitalized in the Reference Hospital of Santa Catarina between April 2010 and April 2011. Species identification was performed using MMSA and PRA-hsp65. These samples were also used to estimate the yield of PCR of 16S rRNA gene, real time PCR, PRA-hsp65 and MMSA, directly of the clinical samples. Results: Among the NTM isolates obtained from Reference Laboratory, M. avium was the most common specie (39,8% of cases), followed by M. fortuitum (14,8%), M. abscessus (12,5%) and M. kansasii (6,8%). Pulmonary cases were more frequent (85,3% of cases) and 61.4% of the isolates were from Grande Florianópolis and Vale do Itajaí. Twenty six cases (34.2%) were characterized as mycobacteriosis. Of these cases, 65.4% were attributed to M. avium Complex and 88.6% were characterized as pulmonary disease. The mean age of patients was 46,1 ± 14,6 years. Men and non-HIV patients were more affected by the disease. At the Reference Hospital, NTM was identified in only one patient with TB diagnosis. PCR and real time PCR showed sensitivity of 91.2% and 100%, respectively. MMSA and PRA-hsp65 identified the species, directly of the sputum, in 50.9% and 47.4% of cases, respectively. When associated, these methods identified the specie in75,4% of samples. Conclusions: This study provided the epidemiology of NTM of Santa Catarina State, that had not been studied yet. It also allowed the assessment of molecular techniques that can be used as tools for early diagnosis and identification of mycobacteria

The performance of prognostic models as predictors of mortality in patients with acute decompensation of cirrhosis

Background. Although several prognostic models have been proposed for cirrhotic patients listed for transplantation, the performance of these scores as predictors of mortality in patients admitted for acute decompensation of cirrhosis has not been satisfactorily investigated.Aims. To study MELD, MELD-Na, MESO, iMELD, Refit-MELD and Refit MELD-Na models as prognostic predictors in cirrhotic patients admitted for acute decompensation, and to compare their performance between admission and 48 hours of hospitalization to predict in-hospital mortality.Material and methods. This cohort study included cirrhotic patients admitted to hospital due to complications of the disease. Individuals were evaluated on admission and after 48 h of hospitalization, and mortality was evaluated during the present admission.Results. One hundred and twenty-three subjects with a mean age of 54.26 ± 10.79 years were included; 76.4% were male. Mean MELD score was 16.43 ± 7.08 and 52.0% of patients were Child-Pugh C. Twenty-seven patients (22.0%) died during hospitalization. Similar areas under the curve (AUROCs) for prognosis of mortality were observed when different models were compared on admission (P > 0.05) and after 48 h of hospitalization (P > 0.05). When models executed after 48 h of hospitalization were compared to their corresponding model calculated on admission, significantly higher AUROCs were obtained for all models (P < 0.05), except for MELD-Na (P = 0.075) and iMELD (P = 0.119).Conclusion. The studied models showed similar accuracy as predictors of in-hospital mortality in cirrhotic patients admitted for acute decompensation. However, the performance of these models was significantly better when applied 48 h after admission when compared to their calculation on admission

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria

Factors associated with 25-hydroxyvitamin D levels in patients with liver cirrhosis

Introduction. Lower 25-hydroxyvitamin D [25(OH)D] levels have been observed in cirrhotic patients and have been related to disease severity. However, most previous studies included patients with very advanced disease, lacking an adequate control for other variables that could interfere with vitamin D levels. We sought to investigate the prevalence of hypovitaminosis D and the factors related to its occurrence.Material and methods. This cross-sectional study included 133 cirrhotic patients and 30 healthy controls. Bivariate and multivariate analyses were performed to determine factors associated with 25(OH)D levels below the lower tertile. Thirty patients who had been recently hospitalized were compared in two time points.Results. Mean 25(OH)D levels were 32.34 ± 11.38 in controls and 27.03 ± 6.22 ng/mL in patients (P = 0.018). 25(OH)D levels were < 30 ng/mL in 69.9% and < 20 ng/mL in 14.3% of the sample. Levels of 25(OH)D below the lower tertile (< 24 ng/mL) were independently associated with higher triceps skinfold and non-Caucasian race. Parathyroid hormone above the reference value (65 pg/mL) was found in 24.6% of patients without association with 25(OH)D or severity of liver disease. Significantly lower levels of 25(OH)D were found at the time of acute decompensation of cirrhosis.Conclusions. In conclusion, hypovitaminosis D was prevalent in cirrhotics and it was associated with adiposity and non-Caucasian race in stable patients with relatively well preserved liver function. However, significantly lower levels were observed during admission for acute decompensation suggesting an impact of systemic inflammation or liver dysfunction on 25(OH)D levels

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

Submitted by Nuzia Santos ([email protected]) on 2015-02-27T12:50:33Z No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-27T12:50:39Z (GMT) No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5)Approved for entry into archive by Nuzia Santos ([email protected]) on 2015-02-27T12:56:22Z (GMT) No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5)Made available in DSpace on 2015-02-27T12:56:22Z (GMT). No. of bitstreams: 1 2014_112.pdf: 385803 bytes, checksum: 73ae8e5baf37fce92c3d713b7f570aed (MD5) Previous issue date: 2014CNPq, LMW and MLBUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Biologia Molecular e Micobactérias. Florianópolis, SC, BrasilUniversidade Federal de Santa Catarina. Laboratório de Protozoologia. Florianópolis, SC, BrasilFundação Oswaldo Cruz. Centro de Pesquisas René Rachou. Laboratório de Imunologia Celular e Molecular. Belo Horizonte, MG, BrasilLaboratório Central do Estado de Santa Catarina. Florianópolis, SC, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Microbiologia. Laboratório de Vírus. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Microbiologia. Laboratório de Vírus. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Instituto de Ciências Biológicas. Departamento de Microbiologia. Laboratório de Vírus. Belo Horizonte, MG, BrasilThe identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteri