Detecção de DNA de micobactérias dos complexos M. tuberculosis e M. avium em amostras parafinadas de pacientes transplantados renais

Introduction: Tuberculosis (TB) is a granulomatous disease caused by Mycobacterium tuberculosis. The genus Mycobacteriumhas two different complexes: M. tuberculosis Complex and M. avium Complex. This is a global health epidemic and remains a major global health problem, besides, the clinical severity of TB is significantly higher in transplanted patients. The detection of these mycobacteria complexes in transplanted patients, by molecular methods, is fundamental for quick treatment of patients and can contribute for rapid and accuracy of diagnosis. Objective: To detect mycobacteria DNA of M. tuberculosis and M. avium Complexes in formalin fixed paraffin-embedded samples (FFPE) of two patients groups: non transplanted and transplanted. Materials and Methods: The study includes 40 FFPE biopsies separated in four groups: NTP – presence of epithelioid granuloma and positive ZN, non-transplanted patients – 9 samples; NTN - presence of epithelioid granuloma and negative ZN, non-transplanted patients – 10 samples; TP – positive ZN, transplanted patients – 9 samples; TN – negative ZN, transplanted patients – 7 samples. Sections were cut for DNA extraction. Samples were submitted to PCR for amplification of: a) β-actin, b) IS6110 insertion and c) IS1245 insertion. DNA evaluation was made by spectrophotometry and efficiency and PCR analysis was made by agarose gels under UV light. Results: In all samples processed, 97.1% were positive for human β-actin gene. In22.2% of NTP group were found the IS6110 insertion sequencebut the IS1245 wasn´t. In the NTN group was not found any sequence. In theTP group, 11.1% of the samples were positive for IS6110 and also 11,1% werepositive for IS1245. In the TN group, 14.3% of the samples were positive forIS6110 and for IS1245, 14.3% was also positive. Conclusion: Although factors such as DNA degradation after formalin fixation and paraffin embedding, were possible to detect DNA from the human gene ...

Busca de fitofármacos com potencial atividade contra Mycobacterium tuberculosis a partir de óleos essenciais

A Tuberculose é um problema grave de Saúde Pública em todo o mundo, causada pelo Mycobacterium tuberculosis e considerada a principal causa de morte entre as doenças infecto-contagiosas. Cerca de 80% da população infectada habitam países subdesenvolvidos, onde o Brasil ocupa a 19ª posição entre os países mais afetados. Embora existam agentes anti-tuberculosos, que são razoavelmente eficazes no tratamento da tuberculose, há uma crescente prevalência de cepas resistentes à multiplos fármacos devido principalmente ao abandono do tratamento.Outros fatores como a co-infecção HIV/TB e a infecção com bactéria em estado de latência denotam a necessidade em se buscar novos fármacos contra TB. Este trabalho teve como objetivo verificar a atividade antimicrobiana do óleo extraído da planta Hedychium coronarium e seus marcadores fitoquímicos frente ao M. tuberculosis utilizando a técnica de Rezasurin Microtiter Assay (REMA), que utiliza como revelador a resazurina. Com a técnica de REMA pudemos verificar a atividade dos óleos essenciais de H. coronarium e seus marcadores fitoquímicos frente ao M. tuberculosis. Futuros estudos buscando o isolamento de compostos a partir desses óleos e testá-los para a verificação de suas atividades anti-tuberculose, é válido e deve ser encorajado, pois existem características antimicrobianas promissoras e é interessante que se realize estudos de sinergismo com outros compostos naturais, podendo assim dar continuidade na busca de novos fitofármacos na luta contra a tuberculose

Obtenção do pró-fármaco recíproco de isoniazida e metronidazol potencialmente ativo contra Mycobacterium tuberculosis

A tuberculose é uma doença que já atingiu milhares de pessoas no mundo e ainda hoje representa uma ameaça à saúde humana, principalmente a partir da descoberta do vírus HIV. Os fármacos disponíveis no mercado não têm suprimido as necessidades, pois exigem longos períodos de tratamento. Este trabalho teve como objetivo o planejamento e a síntese de um novo composto potencialmente ativo para Mycobacterium tuberculosis na forma latente. O planejamento levou em consideração o princípio da latenciação para a obtenção de um pró-fármaco recíproco derivado de isoniazida e metronidazol. O metronidazol, devido à sua ação em bactérias anaeróbicas poderia apresentar atividade nos bacilos que possivelmente sobrevivam durante o processo de necrose caseosa. A obtenção do pró-fármaco foi realizada através da transformação funcional do metronidazol e posterior acoplamento com isoniazida. O trabalho descreve alguns procedimentos e dificuldades encontradas para obtenção do pró-fármaco. Os resultados sugerem a formação do produto, porém, análises de identificação são necessárias para a confirmação

Identificação molecular e genotipagem pela técnica de Spoligotyping de Mycobacterium tuberculosis isolado da população indígena e não-indígena no Mato Grosso do Sul (MS)

O presente trabalho baseou-se na análise de amostras de isolados de M. tuberculosis provenientes da população indígena e não indígena do Mato Grosso do Sul. As análises foram feitas basicamente por três técnicas de Biologia Molecular: PCR-IS6110 e PRA (para identificação) e Spoligotyping (para genotipagem), ressaltando que somente as amostras negativas para PCR-IS6110 foram submetidas ao PRA. Os objetivos do estudo eram: confirmar por técnicas moleculares de PCR e de PRA a identificação do complexo M. tuberculosis e de outras micobactérias, após o descongelamento e cultivo, analisar a incidência de isolados de M. tuberculosis da região do Mato Grosso do Sul que não tem o IS6110, avaliar a incidência de outras micobactérias entre a população indígena e não indígena do Mato Grosso do Sul, agrupar em famílias, por similaridade epidemiológica os isolados de M. tuberculosis procedentes da população indígena e não indígena com TB e avaliar a presença efetiva de grupos genéticos predominantes; Pela técnica de PCR-IS6110, analisamos um total de 119 isolados clínicos, sendo que apenas 6 apresentaram negatividade, sendo assim submetidos à técnica do PRA. Feito isso, também apresentaram resultado positivo para CMTB, nos permitindo concluir que essas técnicas podem se tornar ferramentas de grande valia no diagnóstico rápido da TB. Pela técnica de genotipagem do Spoligotyping foram analisadas 97 amostras e foi constatado um maciço predomínio da família LAM, principalmente da subfamília LAM 9, representados por 4 SITs, sendo SIT 42 com 44 isolados e SITs 177, 1337 e 1075 com respectivamente 4, 2 e 1 isolados cada (essa subfamília representou 52,6% do total de isolados). Outras famílias, tais como T, H e U também estiveram presentes no estudo em menores proporções

Pesquisa de genes de resistência do M. tuberculosis a isoniazida e rifampicina por sequenciamento e pela tecnologia de DNA-STRIP

Tuberculose (TB), causada por Mycobacterium tuberculosis, é uma das doenças infecciosas que mais causam mortes. Estima-se que mais de dois bilhões de pessoas estejam infectadas no mundo. O tratamento da TB consite em associação de fármacos, isoniazida, rifampicina, pirazinamida e etambutol, nos primeiros 2 meses e 4 meses de isoniazida e de rifampicina. Internacionalmente, são consideradas cepas multi resistentes (MDR), as que apresentam resistência simultânea a isoniazida e a rifampicina. A rápida detecção de resistência é essencial para o controle e tratamento da TB, reduzindo, assim, o custo do tratamento e a transmissão da doença. Neste projeto, os isolados já identificados fenotipicamente como resistentes a isoniazida e/ou rifampicina, foram submetidos ao sequenciamento de Sanger para pesquisa de 3 genes relacionados a resistência a isoniazida (katG, inhA e ahpC) e 1 gene de resistência a rifampicina (rpoB). Foi realizada uma comparação destes genes mutados para a resistência utilizando o novo teste desenvolvido pela Biomérieux, denominado GenoType® MTBDRplus, que se baseia na tecnologia DNA-STRIP. Através deste novo teste, foi observada mutação em 22 isolados clínicos de M. tuberculosis para genes de resistência a isoniazida e/ou rifampicina, sendo 4 provenientes do MS e 18 de SP. Já pelo sequenciamento genético foi observada mutação em 24 isolados para genes de resistência a isoniazida e/ou rifampicina, sendo 6 provenientes do MS e 18 de SP. Portanto, através do sequenciamento de Sanger, foi possível detectar um número maior de isolados mutados e mais mutações quando comparado ao teste GenoType® MTBDRplus. Isso acontece porque na técnica de sequenciamento, um fragmento do gene como um todo é analisado e no caso do teste GenoType® MTBDRplus, é verificada apenas a ausência ou presença das mutações mais frequentes descritas na literatura, além de não ser analisado o gene ahpC. A grande ...

Esteroide e Triterpenos de espécies de Qualea: Bioatividade sobre Mycobacterium tuberculosis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Esteroide e Triterpenos de espécies de Qualea: bioatividade sobre Mycobacterium tuberculosis

The chloroform extract of bark of the tropical tree Qualea parviflora (Vochysiaceae) was fractionated by column chromatography on silica gel, yielding triterpenes (lupeol, lupenone, betulin, epi-betulinic acid and friedelin) and a steroid (β-sitosterol). β-sitosterol, lupenone and lupeol were also identified in Q. grandiflora and Q. multiflora, while friedelin was detected only in Q. Multiflora, by means of gas chromatography-mass spectrometry. The anti-Mycobacterium tuberculosis activity of the chloroform extract and isolated compounds was assayed by MABA and MIC values ranged from 250.0 to 31.2 µg/mL. This study is the first to investigate the chemistry and antitubercular activity of apolar compounds from Qualea species.

Bronchoalveolar lavage (BAL) in tapirs (Tapirus terrestris) withMycobacterium tuberculosis

This paper describes the in vivo Bronchoalveolar lavage (BAL)technique by endoscopy in tapirs (Tapirus terrestris) with clinicalsigns of tuberculosis. The technique was performed in two tapirs, male and female,from Curitiba Zoo, Paraná, Brazil. A flexible endoscope and a polyethylene catheterwere used after the chemical restraint of the animals. For BAL technique, 60mL ofsaline 0.9% were infused with a polyethylene catheter, introduced by the endoscope'working channel, and 15mL of BAL were recovered, analyzed and submitted tocytocentrifugation. Slides were stained by Papanicolaou, periodic acid-Schiff (PAS)and Ziehl-Neelsen methods contained high quantity of inflammatory cells on lightmicroscopy (macrophages 27.5%, lymphocytes 0.5%, neutrophis 67% and eosinophis 5%).BAL samples were submitted to culture, bacilloscopy and PCR and were negative forboth animals. Based on this study, it was concluded that the bronchoalveolar lavagetechnique in tapirs is feasible, simple, noninvasive, practical and fast, providingan important clinical information in vivo regarding the functionalstatus of the lower respiratory tract.

Effects of different inspired oxygen fractions on sildenafil-induced pulmonary anti-hypertensive effects in a sheep model of acute pulmonary embolism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ruthenium(II) complexes with hydroxypyridinecarboxylates: screening potential metallodrugs against Mycobacterium tuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Dendrimer nanocarriers for transport modulation across models of the pulmonary epithelium

The purpose of this study was to determine the effect of PEGylation on the interaction of poly(amidoamine) (PAMAM) dendrimer nanocarriers (DNCs) with in vitro and in vivo models of the pulmonary epithelium. Generation-3 PAMAM dendrimers with varying surface densities of PEG 1000 Da were synthesized and characterized. The results revealed that the apical to basolateral transport of DNCs across polarized Calu-3 monolayers increases with an increase in PEG surface density. DNC having the greatest number of PEG groups (n = 25) on their surface traversed at a rate 10-fold greater than its non-PEGylated counterpart, in spite of their larger size. This behavior was attributed to a significant reduction in charge density upon PEGylation. We also observed that PEGylation can be used to modulate cellular internalization. The total uptake of PEG-free DNC into polarized Calu-3 monolayers was 12% (w/w) vs 2% (w/w) for that with 25 PEGs. Polarization is also shown to be of great relevance in studying this in vitro model of the lung epithelium. The rate of absorption of DNCs administered to mice lungs increased dramatically when conjugated with 25 PEG groups, thus supporting the in vitro results. The exposure obtained for the DNC with 25PEG was determined to be very high, with peak plasma concentrations reaching 5 mu gmL(-1) within 3 h. The combined in vitro and in vivo results shown here demonstrate that PEGylation can be potentially used to modulate the internalization and transport of DNCs across the pulmonary epithelium. Modified dendrimers thereby may serve as a valuable platform that can be tailored to target the lung tissue for treating local diseases, or the circulation, using the lung as pathway to the bloodstream, for systemic delivery.

Pulmonary function in obese vs non-obese cats

Obesity is a risk factor in the development of several respiratory diseases. Lung volumes tend to be decreased, especially expiratory reserve volume, increasing expiratory flow limitation during tidal breathing. Barometric whole-body plethysmography is a non-invasive pulmonary function test that allows a dynamic study of breathing patterns. The objective of this study was to compare pulmonary function variables between obese and non-obese cats through the use of barometric whole-body plethysmography. Nine normal-weight and six obese cats were placed in the plethysmograph chamber, and different respiratory variables were measured. There was a significant decrease in tidal volume per kilogram (P=0.003), minute volume per kilogram (P=0.001) and peak inspiratory and expiratory flows per kilogram (P=0.001) in obese cats compared with non-obese cats. Obesity failed to demonstrate a significant increase in bronchoconstriction index variable enhanced pause (Penh), as previously reported in humans and dogs. The results show that feline obesity impairs pulmonary function in cats, although a significant increase in bronchoconstriction indexes was not observed. Non-invasive barometric whole-body plethysmography can help characterise mechanical dysfunction of the airways in obese cats.

Analysis of autonomic modulation after an acute session of resistance exercise at different intensities in chronic obstructive pulmonary disease patients

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Outpatient versus inpatient treatment for acute pulmonary embolism

Background: Pulmonary embolism (PE) is a common life-threatening cardiovascular condition, with an incidence of 23 to 69 new cases per 100,000 people per year. Outpatient treatment instead of traditional inpatient treatment in selected non-high-risk patients with acute PE might provide several advantages, such as reduction of hospitalizations, substantial cost saving and an improvement in health-related quality of life. Objectives: To compare the efficacy and safety of outpatient versus inpatient treatment for acute PE for the outcomes of all-cause and PE-related mortality; bleeding; and adverse events such as hemodynamic instability, recurrence of PE and patients'satisfaction.Search methodsThe Cochrane Peripheral Vascular Diseases Group Trials Search Co-ordinator (TSC) searched the Specialised Register (last searched October 2014) and the Cochrane Central Register of Controlled Trials (CENTRAL; 2014, Issue 9). The TSC also searched clinical trials databases. The review authors searched LILACS (last searched November 2014). Selection criteria: Randomized controlled trials of outpatient versus inpatient treatment in people diagnosed with acute PE. Data collection and analysis: Two review authors selected relevant trials, assessed methodological quality, and extracted and analyzed data. Main results: We included one study, involving 339 participants. We ranked the quality of the evidence as very low due to not blinding the outcome assessors, the small number of events with imprecision in the confidential interval (CI), the small sample size and it was not possible to verify publication bias. For all outcomes, the CIs were wide and included clinically significant treatment effects in both directions: short-term mortality (30 days) (RR 0.33, 95% CI 0.01 to 7.98, P = 0.49), long-term mortality (90 days) (RR 0.98, 95% CI 0.06 to 15.58, P = 0.99), major bleeding at 14 days (RR 4.91, 95% CI 0.24 to 101.57, P = 0.30) and 90 days (RR 6.88, 95% CI 0.36 to 134.14, P = 0.20), recurrent PE within 90 days (RR 2.95, 95% CI 0.12 to 71.85, P = 0.51) and participant satisfaction (RR 0.97, 95% CI 0.92 to 1.03, P = 0.30). PE-related mortality, minor bleeding, and adverse course such as hemodynamic instability and compliance were not assessed by the single included study. Authors' conclusions: Current very low quality evidence from one published randomized controlled trial did not provide sufficient evidence to assess the efficacy and safety of outpatient versus inpatient treatment for acute PE in overall mortality, bleeding and recurrence of PE adequately. Further well-conducted research is required before informed practice decisions can be made.

Pulmonary adenocarcinoma in cattle

The Macroscopic, histological and immunohistochemical aspects of lung acinar adenocarcinoma and the presence of nodules in the abdominal cavity of an adult female bovine are reported. In the necropsy analysis samples were collected from the: lung, heart, spleen, liver, pancreas, kidney, uterus, intestine, brain, and from nodules found in the lung and abdominal cavity, which were routinely processed to be stained by hematoxylin-eosin and for an immunohistochemistry exam with the antibodies: cytokeratin (dilution 1: 200 mu L) and vimentin (dilution 1: 1000 mu L). The histopathological examination revealed neoplastic epithelial cells with acini formation. The immunohistochemical examination of the tumor cells showed positive marking for cytokeratin and the absence of marking for vimentin. According to anatomical, morphological, and histopathological findings, as well as the result of the immunohistochemical examination, the tumor was characterized as lung acinar adenocarcinoma.