Endothelial mediators of 17ß-estradiol-induced coronary vasodilation in the isolated rat heart

The present study was designed to determine relaxation in response to 17ß-estradiol by isolated perfused hearts from intact normotensive male and female rats as well as the contribution of endothelium and its relaxing factors to this action. Baseline coronary perfusion pressure was determined and the vasoactive effects of 17ß-estradiol (10 µM) were assessed by in bolus administration before and after endothelium denudation by infusion of 0.25 µM sodium deoxycholate or perfusion with 100 µM L-NAME, 2.8 µM indomethacin, 0.75 µM clotrimazole, 100 µM L-NAME plus 2.8 µM indomethacin, and 100 µM L-NAME plus 0.75 µM clotrimazole. Baseline coronary perfusion pressure differed significantly between males (84 ± 2 mmHg, N = 61) and females (102 ± 2 mmHg, N = 61). Bolus injection of 10 µM 17ß-estradiol elicited a transient relaxing response in all groups, which was greater in coronary beds from females. For both sexes, the relaxing response to 17ß-estradiol was at least in part endothelium-dependent. In the presence of the nitric oxide synthase inhibitor L-NAME, the relaxing response to 17ß-estradiol was reduced only in females. Nevertheless, in the presence of indomethacin, a cyclooxygenase inhibitor, or clotrimazole, a cytochrome P450 inhibitor, the 17ß-estradiol response was significantly reduced in both groups. In addition, combined treatment with L-NAME plus indomethacin or L-NAME plus clotrimazole also reduced the 17ß-estradiol response in both groups. These results indicate the importance of prostacyclin and endothelium-derived hyperpolarizing factor in the relaxing response to 17ß-estradiol. 17ß-estradiol-induced relaxation may play an important role in the regulation of coronary tone and this may be one of the reasons why estrogen replacement therapy reduces the risk of coronary heart disease in postmenopausal women.

An HPLC-UV method for the measurement of permeability of marker drugs in the Caco-2 cell assay

The Caco-2 cell line has been used as a model to predict the in vitro permeability of the human intestinal barrier. The predictive potential of the assay relies on an appropriate in-house validation of the method. The objective of the present study was to develop a single HPLC-UV method for the identification and quantitation of marker drugs and to determine the suitability of the Caco-2 cell permeability assay. A simple chromatographic method was developed for the simultaneous determination of both passively (propranolol, carbamazepine, acyclovir, and hydrochlorothiazide) and actively transported drugs (vinblastine and verapamil). Separation was achieved on a C18 column with step-gradient elution (acetonitrile and aqueous solution of ammonium acetate, pH 3.0) at a flow rate of 1.0 mL/min and UV detection at 275 nm during the total run time of 35 min. The method was validated and found to be specific, linear, precise, and accurate. This chromatographic system can be readily used on a routine basis and its utilization can be extended to other permeability models. The results obtained in the Caco-2 bi-directional transport experiments confirmed the validity of the assay, given that high and low permeability profiles were identified, and P-glycoprotein functionality was established.

Determining aflatoxins B1, B2, G1 and G2 in maize using florisil clean up with thin layer chromatography and visual and densitometric quantification

A method for determining aflatoxins B1 (AFB1), B2 (AFB2),G1 (AFG1) andG2 (AFG2) in maize with florisil clean up was optimised aiming at one-dimensional thin layer chromatography (TLC) analysis with visual and densitometric quantification. Aflatoxins were extracted with chloroform: water (30:1, v/v), purified through florisil cartridges, separated on TLC plate, detected and quantified by visual and densitometric analysis. The in-house method performance characteristics were determined by using spiked, naturally contaminated maize samples, and certified reference material. The mean recoveries for aflatoxins were 94.2, 81.9, 93.5 and 97.3% in the range of 1.0 to 242 µg/kg for AFB1, 0.3 to 85mg/kg for AFB2, 0.6 to 148mg/kg for AFG1 and 0.6 to 140mg/kg for AFG2, respectively. The correlation values between visual and densitometric analysis for spiked samples were higher than 0.99 for AFB1, AFB2, AFG1 and 0.98 for AFG2. The mean relative standard deviations (RSD) for spiked samples were 16.2, 20.6, 12.8 and 16.9% for AFB1, AFB2, AFG1 and AFG2, respectively. The RSD of the method for naturally contaminated sample (n = 5) was 16.8% for AFB1 and 27.2% for AFB2. The limits of detection of the method (LD) were 0.2, 0.1, 0.1 and 0.1mg/kg and the limits of quantification (LQ) were 1.0, 0.3, 0.6 and 0.6mg/kg for AFB1, AFB2, AFG1 and AFG2, respectively.

In vitro and in vivo antioxidant activity of the pulp of Jatobá-do-cerrado

Abstract Oxygen metabolism in cells causes the production of free radicals, which produce damage, including changes in cell structure and function. Antioxidants are substances that, at low concentrations, slow down or prevent oxidation. Fruits and vegetables contribute to the dietary supply of these compounds. The flora of the Cerrado in Brazil has shown to have high levels of bioactive compounds. This study aimed to characterize the antioxidant activity of the pulp of jatobá-do-cerrado in vitro and in vivo.In vitro antioxidant activity of the aqueous, ethanol and aqueous acetone extracts was evaluated by the DPPH method. We determined total phenols by the Folin-Ciocalteu assay and tannins by the Folin-Denis method.In vivo antioxidant potential of the aqueous acetone extract was evaluated by the TBARS technique. The aqueous acetone extract had the highest antioxidant capacity, followed by the aqueous and ethanol extracts. The same pattern occurred in the extraction of phenols and in the extraction of tannins. In vivo administration of the aqueous acetone extract inhibited lipid peroxidation compared to the control group. The inhibition of peroxidation has increased by elevating the dosage concentration of the extracts, demonstrating a significant antioxidant potential in vivo as well as in vitro.

Are serum cystatin C levels influenced by steroid doses in lupus nephritis patients?

INTRODUCTION: Cystatin C is considered a promising test to evaluate glomerular filtration rate, since it has characteristics of an ideal endogenous marker, being similar or even superior to serum creatinine according to some studies. However, it is possible that some factors (as corticotherapy) could have an influence on serum cystatin C levels regardless of the glomerular filtration rate. The aim of this study was to investigate if different doses of glucocorticoid could have an influence on serum cystatin C levels in lupus nephritis patients. METHODS: We evaluated 42 patients with lupus nephritis that performed 109 different blood collections; their mean age was 37.7 ± 13.1 years old, and 88% were female; the mean estimated glomerular filtration rate was of 61.9 ± 20.0 mL/min. Patients were divided according to their glucocorticoid dose in two groups: A - high (pulse therapy with methylprednisolone and prednisone > 0.5 mg/kg/d, n = 14) versus B - low doses (prednisone ≤ 0.5 mg/kg/d, n = 28). Serum creatinine levels were used as parameters for renal function comparison. Cystatin C was determined by an in-house methodology, using Luminex system flow citometry. RESULTS: Considering these groups, cystatin C levels were different only in the second visit (p = 0.106). But, when the serum creatinine levels were considered in the same groups, a marginally significant difference among them (p = 0.070) was observed, which suggested that the difference in cystatin C levels between the groups was caused by their respective glomerular filtration rate. There was not any difference between those groups that received or did not receive pulse therapy. CONCLUSION: Although some previous studies have shown that glucocorticoid has an influence on serum cystatin C levels, we have not observed such interference in the lupus nephritis patients submitted to corticotherapy.

Inovando na pós-graduação: a experiência do MBA da EAESP/FGV

O artigo aborda a problemática do redesenho dos programas de pós-graduação e analisa as várias questões que vêm sendo discutidas neste campo: o desenvolvimento de cursos alternativos de pós-graduação, a redução do número de créditos do mestrado e o enxugamento dos programas deste nível, com vistas à diminuição do tempo médio de duração dos cursos. Sob esta perspectiva, é apresentada a experiência da EAESP/FGV (Escola de Administração de Empresas de São Paulo, da Fundação Getúlio Vargas) no desenvolvimento do MBA (Master in Business Administration), experiência pioneira que vem ao encontro da necessidade de inovações na pós-graduação em Administração.

O fenômeno dos MPAs brasileiros: hibridismo, diversidade e tensões

Na década de 1990, os programas de MBA - Master in Business Administration - ganharam sólida reputação e expandiram-se para além das fronteiras norte-americanas. No Brasil, o movimento ganhou força com o crescimento vigoroso do ensino profissionalizante de gestão. Este artigo tem como objetivo apresentar e analisar, sob uma perspectiva crítica, o fenômeno dos Mestrados Profissionais em Administração (MPAs) brasileiros. Para traçar um retrato do fenômeno, realizamos pesquisa bibliográfica e documental, e entrevistas com coordenadores e pessoas-chave dos principais programas locais. Apesar de eventualmente classificados como MBAs, os MPAs apresentam características que os diferenciam do modelo norteamericano. Ao buscar responder ao contexto socioeconômico e às restrições regionais, cada um deles seguiu rotas próprias, o que gerou um quadro marcado pela diversidade. Acreditamos que o fenômeno que este artigo descreve e analisa possa contribuir para o debate em curso sobre o ensino de Administração.

Transparency of the municipal public management: a study from the homepages of the large Brazilian municipalities

This study attempts to check the transparency level of information in public administration published in the homepages of 96 municipalities included among the 100 most populous in Brazil and what characteristics and socioeconomic indicators of the municipalities can contribute to explain the level of transparency observed. The level of transparency in public administration was established from a research model called Transparency Index Municipal Public Management (ITGP-M) constructed based on international codes of good governance and transparency, the Brazilian legislation and the experiences of previous studies of similar nature conducted in Brazil and abroad. The empirical evidence point to low levels of transparency, incompatible with the level of socioeconomic development of municipalities. Moreover, we can conclude that, overall, there is an association between the socioeconomic conditions of the municipalities and the levels of transparency in the disclosure of information about public administration observed in sites of large municipalities as in this study.

An indirect immunofluorescence assay to detect antibodies against St. Louis Encephalitis virus

An in house indirect immmunofluorescence assay ( IFA ) in relation to neutralization (NT) reference test, was assessed as a fast and cheap method to carry out serological surveys for St. Louis Encephalitis virus (SLE). Sera obtained from 213 blood donors were analyzed by both tests. The prevalence of seropositivity obtained with IFA was lower than (30.98%) that observed on NT (41.78%). The relative specificity rate of IFA was 96.77% whereas its relative sensitivity rate was 69.66%. Kappa index showed a good correlation between both tests. The results indicate that neutralization assay is still the serological test with the highest sensitivity and specificity relative rates for detecting antibodies against SLE virus. Nevertheless, the IFA could be useful as an alternative test in order to learn the circulation of the Flavivirus genus in a certain area.

Replacement of HIV p24 Ag test by a multiplex RT-PCR method for primary screening of blood donors

Nucleic Acid Testing (NAT) as a tool for primary screening of blood donors became a reality in the end of the 1990 decade. We report here the development of an "in-house" RT-PCR method that allows the simultaneous (multiplex) detection of HCV and HIV-RNA in addition to an artificial RNA employed as an external control. This method detects all HIV group M subtypes, plus group N and O, with a detection threshold of 500 IU/mL. After validation, the method replaced p24 Ag testing, in use for blood donation screening since 1996 at our services. From July 2001 to February 2006, 102,469 donations were tested and 41 (0.04%) were found HIV-RNA reactive. One NAT-only reactive donation (antibody non-reactive) was observed, with subsequent seroconversion of the implied donor, giving a yield of 1:102,469. This rate is in contrast to the international experience that reports a detection of approximately 1:600,000 - 1:3,100,000 of isolated HIV-RNA donations.

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

Evidência sorológica da infecção aguda pelo Toxoplasma gondii em gestantes de Cascavel, Paraná

Para verificar a ocorrência da toxoplasmose em Cascavel, Paraná, cidade próxima a região onde ocorreu o maior surto epidêmico descrito mundialmente, 334 amostras de soros de gestantes foram triadas pelo ensaio imunoenzimático comercial IgG no Laboratório Municipal de Cascavel, e confirmadas no Instituto de Medicina Tropical de São Paulo por imunofluorescência IgG, ensaio imunoenzimático e avidez de IgG in house. A soropositividade pelo IgG comercial foi 54,2%, pela imunofluorescência 54,8% e pelo IgG in house 53,9%, com boa concordância entre imunofluorescência/IgG comercial (Kappa=0,963781; co-positividade=97,8%; co-negatividade=98,7%) e imunofluorescência/IgG in house (Kappa=0,975857; co-positividade=97,8%; co-negatividade=100%). A evidência de infecção aguda nas gestantes foi similar tanto pela avidez de IgG (2,4% ao ano) como pela análise estatística de tendência (teste χ2) por faixa etária (2% ao ano), sugerindo que a triagem sorológica pré-natal e a vigilância epidemiológica são imprescindíveis para redução do risco da toxoplasmose na região, embora sem evidência de surto epidêmico.

Extração de DNA a partir de sangue humano coagulado para aplicação nas técnicas de genotipagem de antígenos leucocitários humanos e de receptores semelhantes à imunoglobulina

O objetivo deste estudo foi padronizar uma metodologia de extração de DNA de alta qualidade a partir de amostras de sangue coagulado. Quarenta e oito amostras de sangue humano coagulado foram utilizadas para a extração de DNA pelo kit comercial EZ-DNA® (Biological Industries, Beit Haemek, Israel), pelo kit de coluna Neoscience® (One Lambda Inc., San Diego, CA) e pelo método modificado de salting out. Apenas o método de salting out foi capaz de extrair altas concentrações de DNA (média, 180ng/µL), as quais foram medidas pelo detector de fluorescência Qubit® (Invitrogen, USA). Este método permitiu a amplificação dos genes HLA (human leukocyte antigens) pela tecnologia PCR-SSO (polymerase chain reaction - specific sequence of oligonucleotides) Luminex, a qual exige DNA de boa qualidade, e de genes KIR (killer cell immunoglobulin-like receptors) pela técnica made in house PCR-SSP (polymerase chain reaction-sequence specific of primers), a qual demanda uma concentração específica de DNA (10ng/µL). Concluímos que a técnica de salting out modificada foi muito eficiente, simples e rápida para a extração de DNA de amostras de sangue humano coagulado, com o objetivo de realizar a genotipagem de genes HLA e KIR.

Hibridização reversa e sequenciamento na genotipagem do vírus da hepatite C

INTRODUÇÃO: Os métodos de genotipagem do vírus da hepatite C têm sido muito discutidos. O objetivo deste trabalho foi comparar as metodologias de hibridização reversa e sequenciamento direto para a genotipagem do vírus da hepatite C. MÉTODOS: Noventa e uma amostras de plasma de pacientes assistidos na Faculdade de Medicina de Botucatu da Universidade Estadual Paulista foram utilizadas. A genotipagem por hibridização reversa foi realizada utilizando o kit comercial INNO-LiPA® v.1.0. O sequenciamento direto foi efetuado em sequenciador automático utilizando protocolos in house. RESULTADOS: A genotipagem por sequenciamento direto mostrou-se eficiente na resolução dos resultados inconclusivos pelo kit comercial. O kit mostrou resultados errôneos em relação à subtipagem viral. Além disso, a genotipagem por sequenciamento direto revelou um erro do kit com relação à determinação genotípica questionando a eficiência do método também para a identificação do genótipo viral. CONCLUSÕES: A genotipagem realizada por meio de sequenciamento direto permite uma maior acurácia na classificação viral quando comparada à hibridização reversa.

Diagnosing schistosomiasis: where are we?

In light of the World Health Organization's initiative to extend schistosomiasis morbidity and mortality control programs by including a disease elimination strategy in low endemic settings, this paper reviews diagnostic tools described during the last decades and provide an overview of ongoing efforts in making an efficient diagnostic tool available worldwide. A literature search on PubMed using the search criteria schistosomiasis and diagnosis within the period from 1978 to 2013 was carried out. Articles with abstract in English and that used laboratory techniques specifically developed for the detection of schistosomiasis in humans were included. Publications were categorized according to the methodology applied (parasitological, immunological, or molecular) and stage of development (in house development, limited field, or large scale field testing). The initial research generated 4,535 publications, of which only 643 met the inclusion criteria. The vast majority (537) of the publications focused on immunological techniques; 81 focused on parasitological diagnosis, and 25 focused on molecular diagnostic methods. Regarding the stage of development, 307 papers referred to in-house development, 202 referred to limited field tests, and 134 referred to large scale field testing. The data obtained show that promising new diagnostic tools, especially for Schistosoma antigen and deoxyribonucleic acid (DNA) detection, which are characterized by high sensitivity and specificity, are being developed. In combination with international funding initiatives these tools may result in a significant step forward in successful disease elimination and surveillance, which is to make efficient tests accessible and its large use self-sustainable for control programs in endemic countries.