Potencial terapêutico do extrato de própolis na sepse experimental

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Medicina, Programa de Pós-Graduação em Ciências Médicas, 2014.A sepse é uma resposta inflamatória sistêmica e é uma das maiores causas de morbidade e mortalidade em Unidade de Terapia Intensiva. Nos últimos anos, ainda que com muitas descobertas no campo da antibioticoterapia, somente alguns poucos benefícios foram observados em pacientes sépticos. A flora brasileira é um dos maiores investimentos de companhias farmacêuticas em busca de terapias alternativas. A própolis, por exemplo, é uma dessas terapias devido as suas propriedades antimicrobianas. Visto que nosso país é rico nesta fonte natural, este estudo foi conduzido para avaliar os efeitos do extrato de própolis verde nas alterações comportamentais e cognitivas decorrentes da infecção sistêmica. Com o objetivo de avaliar esta proposta, foram usados 40 ratos Wistar machos, com 2,5 meses de idade, anestesiados com ketamina e xilazina antes dos procedimentos cirúrgicos. Os animais controles (operação fictícia – OF) não receberam perfuração e nem ligadura. Metade dos animais foram administrados com os antibióticos clindamicina (25 mg/kg, i.p.) + gentamicina (3 mg/kg, i.p.) antes dos procedimentos cirúrgicos, e novamente com 24 e 48 horas.O extrato de própolis (100 mg/kg, i.p.) foi administrado no mesmo regime de tratamento dos antibióticos. As avaliações comportamentais e cognitivas foram avaliadas com 48 e 72h. Para tal, eles foram divididos em 4 grupos: 1) OF+antibióticos, 2) OF+própolis, 3) Sepse+antibióticos, e 4) Sepse+própolis, para serem avaliados no teste do campo aberto (locomoção), labirinto em cruz elevado (ansiedade), nado forçado (depressão/estresse), e esquiva inibitória (memória). Foi observado que os animais que sobreviveram à sepse tratados com antibióticos diminuíram o percentual de entradas nos braços abertos e tempo de permanência nesses braços do LCE, resposta sugestiva de aumento de níveis de ansiedade. Nesses mesmos animais, não foi observada qualquer alteração na locomoção, enquanto que no teste do nado forçado eles reduziram o tempo de imobilidade, sugerindo comportamento de estresse resultante da reação de fuga/luta. Além disso, foi observado também prejuízo na memória de curta e longa duração, que foi revertido pelo extrato de própolis, mantendo uma resposta similar aos animais controles OF. Em um contexto geral, o extrato de própolis pode prevenir as alterações comportamentais e cognitivas resultantes da sepse experimental, cabendo investigar os possíveis mecanismos celulares e moleculares envolvidos com as respostas dessa terapia natural. ______________________________________________________________________________ ABSTRACTSepsis is a systemic inflammatory response and it is on the major cause of morbidity and mortality in the intensive care unit. In recent years, even with many discoveries in the field of antibiotic therapy, only a few clinical benefits were observed in septic patients. The Brazilian flora is one of the biggest investments of pharmaceutical companies seeking alternative therapies. Propolis, for example, is one of these therapies because has antimicrobial properties. Given that our country is rich in these natural resources, this study sought to evaluate the effects of green propolis in behavioral and cognitive impairments resulting from systemic infection. In order to evaluate this proposal, it was used 40 Wistar male rats, 2.5 months old, anesthetized with ketamine + xylazine before the surgical procedure. The control animals (sham) did not receive neither ligation and perforation. Half of the animals were administered with the antibiotics clindamicine (25 mg/kg, i.p.) + gentamicin (3 mg/kg, i.p.) before surgery procedures, and again 24h and 48h latter. Propolis extract (100 mg/kg, i.p.) was administered in the same treatment regimen of the antibiotics. The behavioral and cognitive assessments were evaluated with 48h or 72h latter. They were divided into 4 groups: 1) Sham+antibiotics, 2) Sham+propolis, 3) Sepsis+antibiotics, and 4) Sepsis+propolis, which were evaluated in the open field (locomotion), elevated plus maze-EPM (anxiety), forced swimming (depression) and step down inhibitory avoidance (memory) tests. It was observed that the animals subjected to systemic infection decreased the percentage of open arms entries and open arms time of the EPM, suggestive of anxiety-like behavior. No changes were observed in locomotion, whereas in the forced swimming test animals that survived the infection showed a reduced immobility time, suggesting stress-like behavior resulted from the reaction of flight/fight. Systemic infection survivor rats showed impairment in short and long term memories, which was blocked by propolis extract with a similar response of the sham surgery.In overall, propolis extract can prevent behavioral and cognitive changes resulting from experimental sepsis, leaving to investigate the cellular and molecular mechanisms involved in these natural therapy responses

Modulation of the immune response by Fonsecaea pedrosoi morphotypes in the course of experimental chromoblastomycosis and their role on inflammatory response chronicity

A common theme across multiple fungal pathogens is their ability to impair the establishment of a protective immune response. Although early inflammation is beneficial in containing the infection, an uncontrolled inflammatory response is detrimental and may eventually oppose disease eradication. Chromoblastomycosis (CBM), a cutaneous and subcutaneous mycosis, caused by dematiaceous fungi, is capable of inducing a chronic inflammatory response. Muriform cells, the parasitic form of Fonsecaea pedrosoi, are highly prevalent in infected tissues, especially in long-standing lesions. In this study we show that hyphae and muriform cells are able to establish a murine CBM with skin lesions and histopathological aspects similar to that found in humans, with muriform cells being the most persistent fungal form, whereas mice infected with conidia do not reach the chronic phase of the disease. Moreover, in injured tissue the presence of hyphae and especially muriform cells, but not conidia, is correlated with intense production of pro-inflammatory cytokines in vivo. Highthroughput RNA sequencing analysis (RNA-Seq) performed at early time points showed a strong up-regulation of genes related to fungal recognition, cell migration, inflammation, apoptosis and phagocytosis in macrophages exposed in vitro to muriform cells, but not conidia. We also demonstrate that only muriform cells required FcγR and Dectin-1 recognition to be internalized in vitro, and this is the main fungal form responsible for the intense inflammatory pattern observed in CBM, clarifying the chronic inflammatory reaction observed in most patients. Furthermore, our findings reveal two different fungal-host interaction strategies according to fungal morphotype, highlighting fungal dimorphism as an important key in understanding the bipolar nature of inflammatory response in fungal infections

Variabilidade de genótipos de milho quanto à composição de carotenoides nos grãos

The objective of this work was to characterize 134 maize (Zea mays) genotypes, for carotenoids content and build a genetic profile to facilitate future breeding to increase grain nutritional value (biofortification). Seeds came from experimental fields and from the Banco Ativo de Germoplasma of Embrapa Milho e Sorgo, Sete Lagoas, MG, Brazil. The genotypes were commercial hybrids, varieties, and inbred lines developed by the Embrapa biofortification program and other accessions, chosen for their yellow-orange endosperm color. Total grain carotenoids, carotenes, xanthophylls (monohydroxylates and dihydroxylates) were determined by chromatographic-spectrophotometric methods. The detected averages were: 22.34 µg g-1 for total carotenoids, 2.55 µg g-1 for carotenes, 3.86 μgág-1 for monohydroxylated xanthophylls, and 15.93 µg g-1 for dihydroxylated xanthophylls. The genotypes variability was divided into 18 groups by the method of Tocher. The Embrapa germplasm has variability and potential for development of maize lines biofortified for total of provitamin A carotenoids.O objetivo deste trabalho foi caracterizar e avaliar a variabilidade quanto ao teor e perfil de carotenoides nos grãos de 134 genótipos de milho (Zea mays), com vistas à utilização em programas de biofortificação. Os materiais foram provenientes dos campos experimentais e do Banco Ativo de Germoplasma da Embrapa Milho e Sorgo, Sete Lagoas, MG. São cultivares e híbridos comerciais, linhagens-elite e outros acessos escolhidos com base na coloração amarelo-alaranjada do endosperma. A quantificação do teor de carotenoides totais, carotenos e xantofilas mono-hidroxilada e di-hidroxilada dos grãos foi realizada por método cromatográfico-espectrofotométrico. As médias encontradas nos grãos foram 22,34 µg g-1 de carotenoides totais, 2,55 µg g-1 de carotenos, 3,86 µg g-1 de xantofilas mono-hidroxiladas e 15,93 µg g-1 de xantofilas di-hidroxiladas. Os genótipos foram agrupados em 18 grupos pelo método de Tocher. O germoplasma da Embrapa possui potencial para ser usado em programas de desenvolvimento de linhagens de milho biofortificadas, quanto ao total de carotenoides pró-vitamina A

Anti‑EpCAM antibodies for detection of metastatic carcinoma in effusions and peritoneal wash

Epithelial cell adhesion molecule (EpCAM) has been used as diagnostic/prognostic marker and therapeutic target. The aim of the present study was to compare immunoreactivity of antibodies against distinct epitopes in the ectodomain of EpCAM for detection of carcinoma from different primary sites and of different histological types in effusions and peritoneal wash. Two antibodies against epitopes in the EGF‑like domain I (clones Moc‑31 and Ber‑EP4) and one antibody against the epitope in the cysteine‑poor region (158210) of EpCAM were used (all commercially available). Independently of the clone used, EpCAM overexpression was observed in almost all samples when all the adenocarcinoma samples were analyzed together. By using Moc‑31, EpCAM overexpression was observed in all samples of adenocarcinoma. Absence of EpCAM overexpression was observed in a few adenocarcinoma samples at some sites of tumor origin, including ovary, breast and stomach, when Ber‑EP4 and 158210 were used. Regarding carcinomas aside from adenocarcinomas, histological types, such as squamous cell, urothelial and small cell carcinoma showed different degrees of EpCAM expression according to the antibody used. In squamous cell carcinoma, overexpression was observed only with the clone 158210. It was concluded that, overall, most samples of metastatic carcinoma from effusions showed overexpression of EpCAM. However, there are significant variations in its detection according to the primary site, histological type of the carcinoma and depending on the antibody used. Thus, the use of more than one type of anti‑EpCAM antibody would increase the chance of its detection in metastatic carcinoma effusion