Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Fludarabine induces apoptosis in chronic lymphocytic leukemia - the role of P53, Bcl-2, Bax, Mcl-1, and Bag-1 proteins

The expression of P53, Bcl-2, Bax, Bag-1, and Mcl-1 proteins in CD5/CD20-positive B-chronic lymphocytic leukemia (B-CLL) cells from 30 typical CLL patients was evaluated before and after 48 h of incubation with 10-6 M fludarabine using multiparametric flow cytometric analysis. Protein expression was correlated with annexin V expression, Rai modified clinical staging, lymphocyte doubling time, and previous treatment. Our main goal was to determine the predictive value of these proteins in CLL cells in terms of disease evolution. Bcl-2 expression decreased from a median fluorescence index (MFI) of 331.71 ± 42.2 to 245.81 ± 52.2 (P < 0.001) after fludarabine treatment, but there was no difference between viable cells (331.57 ± 44.6 MFI) and apoptotic cells (331.71 ± 42.2 MFI) before incubation (P = 0.859). Bax expression was higher in viable cells (156.24 ± 32.2 MFI) than in apoptotic cells (133.56 ± 35.7 MFI) before incubation, probably reflecting defective apoptosis in CLL (P = 0.001). Mcl-1 expression was increased in fludarabine-resistant cells and seemed to be a remarkable protein for the inhibition of the apoptotic process in CLL (from 233.59 ± 29.8 to 252.04 ± 35.5; P = 0.033). After fludarabine treatment, Bag-1 expression was increased in fludarabine-resistant cells (from 425.55 ± 39.3 to 447.49 ± 34.5 MFI, P = 0.012), and interestingly, this higher expression occurred in patients who had a short lymphocyte doubling time (P = 0.022). Therefore, we could assume that Bag-1 expression in such situation might identify CLL patients who will need treatment earlier.

Stimulated mast cells promote maturation of myocardial microvascular endothelial cell neovessels by modulating the angiopoietin-Tie-2 signaling pathway

Angiopoietin (Ang)-1 and Ang-2 interact in angiogenesis to activate the Tie-2 receptor, which may be involved in new vessel maturation and regression. Mast cells (MCs) are also involved in formation of new blood vessels and angiogenesis. The present study was designed to test whether MCs can mediate angiogenesis in myocardial microvascular endothelial cells (MMVECs). Using a rat MMVEC and MC co-culture system, we observed that Ang-1 protein levels were very low even though its mRNA levels were increased by MCs. Interestingly, MCs were able to enhance migration, proliferation, and capillary-like tube formation, which were associated with suppressed Ang-2 protein expression, but not Tie-2 expression levels. These MCs induced effects that could be reversed by either tryptase inhibitor [N-tosyl-L-lysine chloromethyl ketone (TLCK)] or chymase inhibitor (N-tosyl-L-phenylalanyl chloromethyl ketone), with TLCK showing greater effects. In conclusion, our data indicated that MCs can interrupt neovessel maturation via suppression of the Ang-2/Tie-2 signaling pathway.

Adrenomedullin and adrenotensin regulate collagen synthesis and proliferation in pulmonary arterial smooth muscle cells

To understand the pathophysiological mechanisms of pulmonary arterial smooth muscle cell (PASMC) proliferation and extracellular-matrix accumulation in the development of pulmonary hypertension and remodeling, this study determined the effects of different doses of adrenomedullin (ADM) and adrenotensin (ADT) on PASMC proliferation and collagen synthesis. The objective was to investigate whether extracellular signal-regulated kinase (ERK1/2) signaling was involved in ADM- and ADT-stimulated proliferation of PASMCs in 4-week-old male Wistar rats (body weight: 100-150 g, n=10). The proliferation of PASMCs was examined by 5-bromo-2-deoxyuridine incorporation. A cell growth curve was generated by the Cell Counting Kit-8 method. Expression of collagen I, collagen III, and phosphorylated ERK1/2 (p-ERK1/2) was evaluated by immunofluorescence. The effects of different concentrations of ADM and ADT on collagen I, collagen III, and p-ERK1/2 protein expression were determined by immunoblotting. We also investigated the effect of PD98059 inhibition on the expression of p-ERK1/2 protein by immunoblotting. ADM dose-dependently decreased cell proliferation, whereas ADT dose-dependently increased it; and ADM and ADT inhibited each other with respect to their effects on the proliferation of PASMCs. Consistent with these results, the expression of collagen I, collagen III, and p-ERK1/2 in rat PASMCs decreased after exposure to ADM but was upregulated after exposure to ADT. PD98059 significantly inhibited the downregulation by ADM and the upregulation by ADT of p-ERK1/2 expression. We conclude that ADM inhibited, and ADT stimulated, ERK1/2 signaling in rat PASMCs to regulate cell proliferation and collagen expression.

Propofol inhibits burn injury-induced hyperpermeability through an apoptotic signal pathway in microvascular endothelial cells

Recent studies have revealed that an intrinsic apoptotic signaling cascade is involved in vascular hyperpermeability and endothelial barrier dysfunction. Propofol (2,6-diisopropylphenol) has also been reported to inhibit apoptotic signaling by regulating mitochondrial permeability transition pore (mPTP) opening and caspase-3 activation. Here, we investigated whether propofol could alleviate burn serum-induced endothelial hyperpermeability through the inhibition of the intrinsic apoptotic signaling cascade. Rat lung microvascular endothelial cells (RLMVECs) were pretreated with propofol at various concentrations, followed by stimulation with burn serum, obtained from burn-injury rats. Monolayer permeability was determined by transendothelial electrical resistance. Mitochondrial release of cytochrome C was measured by ELISA. Bax and Bcl-2 expression and mitochondrial release of second mitochondrial-derived activator of caspases (smac) were detected by Western blotting. Caspase-3 activity was assessed by fluorometric assay; mitochondrial membrane potential (Δψm) was determined with JC-1 (a potential-sensitive fluorescent dye). Intracellular ATP content was assayed using a commercial kit, and reactive oxygen species (ROS) were measured by dichlorodihydrofluorescein diacetate (DCFH-DA). Burn serum significantly increased monolayer permeability (P<0.05), and this effect could be inhibited by propofol (P<0.05). Compared with a sham treatment group, intrinsic apoptotic signaling activation - indicated by Bax overexpression, Bcl-2 downregulation, Δψm reduction, decreased intracellular ATP level, increased cytosolic cytochrome C and smac, and caspase-3 activation - was observed in the vehicle group. Propofol not only attenuated these alterations (P<0.05 for all), but also significantly decreased burn-induced ROS production (P<0.05). Propofol attenuated burn-induced RLMVEC monolayer hyperpermeability by regulating the intrinsic apoptotic signaling pathway.

Diabetes mellitus affects the biomechanical function of the callus and the expression of TGF-beta1 and BMP2 in an early stage of fracture healing

Transforming growth factor beta 1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) are important regulators of bone repair and regeneration. In this study, we examined whether TGF-β1 and BMP-2 expressions were delayed during bone healing in type 1 diabetes mellitus. Tibial fractures were created in 95 diabetic and 95 control adult male Wistar rats of 10 weeks of age. At 1, 2, 3, 4, and 5 weeks after fracture induction, five rats were sacrificed from each group. The expressions of TGF-β1 and BMP2 in the fractured tibias were measured by immunohistochemistry and quantitative reverse-transcription polymerase chain reaction, weekly for the first 5 weeks post-fracture. Mechanical parameters (bending rigidity, torsional rigidity, destruction torque) of the healing bones were also assessed at 3, 4, and 5 weeks post-fracture, after the rats were sacrificed. The bending rigidity, torsional rigidity and destruction torque of the two groups increased continuously during the healing process. The diabetes group had lower mean values for bending rigidity, torsional rigidity and destruction torque compared with the control group (P<0.05). TGF-β1 and BMP-2 expression were significantly lower (P<0.05) in the control group than in the diabetes group at postoperative weeks 1, 2, and 3. Peak levels of TGF-β1 and BMP-2 expression were delayed by 1 week in the diabetes group compared with the control group. Our results demonstrate that there was a delayed recovery in the biomechanical function of the fractured bones in diabetic rats. This delay may be associated with a delayed expression of the growth factors TGF-β1 and BMP-2.

Estabilidade do suco tropical de acerola (Malpighia emarginata d.c.) adoçado envasado pelos processos hot-fill e asséptico

Este trabalho objetivou avaliar a estabilidade do suco tropical de acerola adoçado, elaborado pelos processos hot fill (garrafas de vidro) e asséptico (embalagens cartonadas), com relação às alterações químicas e físico-químicas (pH, sólidos solúveis totais, acidez total titulável, cor, açúcares redutores, não redutores e totais), sensoriais e microbiológicas, durante 350 dias de armazenamento em condições similares às de comercialização (28 °C ± 2 °C). Ao final do experimento, constatou-se que as amostras de suco de ambos os processos mantiveram uma adequada estabilidade microbiológica. O suco do processo hot fill teve maior aceitação global, enquanto o do processo asséptico manteve, ao final dos 350 dias, a aceitação inicial. As amostras do processo asséptico apresentaram inicialmente melhor sabor em comparação com as do processo hot fill, no entanto, as do processo hot fill mantiveram o sabor estável, enquanto o sabor do suco do processo asséptico teve menor aceitação ao longo do armazenamento. Ainda foram observadas, alterações químicas e físico-químicas nos sucos de ambos os processos. Em geral, o processo hot fill foi o mais eficiente em manter a estabilidade do suco.

Hospitalizações por condições cardiovasculares sensíveis à atenção primária em municípios goianos

OBJETIVO: Analisar taxas de hospitalização por condições cardiovasculares sensíveis à atenção primária. MÉTODOS: Estudo ecológico com 237 municípios do Estado de Goiás, de 2000 a 2008, utilizando dados do Sistema de Informação Hospitalar e Sistema de Informação da Atenção Básica. As taxas de hospitalização foram calculadas pela proporção entre o número de hospitalizações por condições cardiovasculares e a população acima de 40 anos. Foram avaliadas em triênios: A (2000-2002), B (2003-2005) e C (2006-2008), segundo sexo, faixa etária, porte populacional, pertencimento à região metropolitana, macrorregião de saúde, distância da capital, Índice de Condições de Vida e Saúde e cobertura de Estratégia Saúde da Família. A cobertura populacional potencial da Saúde da Família foi calculada conforme diretrizes do Ministério da Saúde. A variabilidade das taxas foi avaliada segundo teste t e ANOVA. RESULTADOS: Ocorreram 253.254 internações (17,2% do total) por condições cardiovasculares sensíveis à atenção primária. As taxas de hospitalização diminuíram entre os triênios: A (213,5, dp = 104,6), B (199,7, dp = 96,3) e C (150,2, dp = 76,1), com diferença entre os períodos A-C e B-C (p < 0,001). Porte populacional municipal não influenciou o comportamento das taxas. Municípios próximos à capital e aqueles da região metropolitana apresentaram maiores taxas (p < 0,001). Em todos os percentis do Índice de Condições de Vida e Saúde, houve redução das taxas (p < 0,001), exceto no percentil 1. Redução foi também observada em todas as macrorregiões, exceto na região nordeste do estado. A redução das taxas ocorreu independentemente da cobertura da Saúde da Família. CONCLUSÕES: As taxas de hospitalização por condições cardiovasculares sensíveis à atenção primária diminuíram nesses municípios, independentemente da cobertura da Saúde da Família.

Candida esophagitis: species distribution and risk factors for infection

Although Candida albicans is the main cause of fungal esophagitis, other species such as C. tropicalis, C. krusei and C. stellatoidea have also been implicated. Several studies have identified risk factors for C. albicans esophagitis. However, data for non-C. albicans species is still sparse. The aim of this study was to determine the etiology of Candida esophagitis in our medical centre over an 18-month period. Additionally, we aimed to investigate predisposing conditions for esophageal candidosis caused by different Candida species. A total of 21,248 upper gastroscopies were performed in Santa Casa Complexo Hospitalar between January 2005 and July 2006. The prevalence of Candida esophagitis was 0.74% (n = 158). C. albicans caused the vast majority of infections (96.2%), followed by C. tropicalis (2.5%), C. lusitaniae (0.6%) and C. glabrata (0.6%). There were 81 women (51.3%) and 77 men (48.7%). No case of mixed infection occurred. Concomitant oral candidosis was documented for 10.8% (n = 17). Most of cases (55.1%) involved outpatients. Around one fifth of patients in our cohort had no identifiable risk factors for esophageal candidosis (20.8%). Since nearly all infections were caused by C. albicans we were not able to determine risk factors for esophagitis caused by other Candida species.

Genetic diversity and primary resistance among HIV-1-positive patients from Maringá, Paraná, Brazil

The objective of this study is to identify subtypes of Human Immunodeficiency Virus type 1 (HIV-1) and to analyze the presence of mutations associated to antiretroviral resistance in the protease (PR) and reverse transcriptase (RT) regions from 48 HIV-1 positive treatment naïve patients from an outpatient clinic in Maringá, Paraná, Brazil. Sequencing was conducted using PR, partial RT and group-specific antigen gene (gag) nested PCR products from retrotranscribed RNA. Transmitted resistance was determined according to the Surveillance Drug Resistance Mutation List (SDRM) algorithm. Phylogenetic and SimPlot analysis of concatenated genetic segments classified sequences as subtype B 19/48 (39.6%), subtype C 12/48 (25%), subtype F 4/48 (8.3%), with 13/48 (27.1%) recombinant forms. Most recombinant forms were B mosaics (B/F 12.5%, B/C 10.4%), with one C/F (2.1%) and one complex B/C/F mosaic (2.1%). Low levels of transmitted resistance were found in this study, 2/48 (2.1% to NRTIs and 2.1% for PI). This preliminary data may subsidize the monitoring of the HIV evolution in the region.

FUNGEMIA CAUSED BY Candida SPECIES IN A CHILDREN'S PUBLIC HOSPITAL IN THE CITY OF SÃO PAULO, BRAZIL: STUDY IN THE PERIOD 2007-2010

Candidemia remains a major cause of morbidity and mortality in the health care environment. The epidemiology of Candida infection is changing, mainly in relation to the number of episodes caused by species C. non-albicans. The overall objective of this study was to evaluate the frequency of yeasts of the genus Candida, in a four-year period, isolated from blood of pediatric patients hospitalized in a public hospital of the city of São Paulo, Brazil. In this period, yeasts from blood of 104 patients were isolated and, the identified species of Candida by phenotypic and genotypic methods were: C. albicans (39/104), C. tropicalis (25/104), C. parapsilosis (23/104), Pichia anomala (6/104), C. guilliermondii (5/104), C. krusei (3/104), C. glabrata (2/104) and C. pararugosa (1/104). During the period of the study, a higher frequency of isolates of C. non-albicans (63.55%) (p = 0.0286) was verified. In this study we verified the increase of the non-albicans species throughout the years (mainly in 2009 and 2010). Thus, considering the peculiarities presented by Candida species, a correct identification of species is recommended to lead to a faster diagnosis and an efficient treatment.

Trypanocide treatment among adults with chronic Chagas disease living in Santa Fe city (Argentina), over a mean follow-up of 21 years: parasitological, serological and clinical evolution

The efficacy of treatment with nifurtimox and/or benznidazole among adults with chronic Chagas disease with no previous electrocardiographic disturbances was evaluated over a mean follow-up of 21 years, by means of conventional serology, xenodiagnosis, clinical examination, electrocardiograms and chest X-ray. One hundred and eleven patients, between 17 and 46 years old, were studied: 54 underwent treatment (nifurtimox 27, benznidazole 27) and 57 remained untreated (control group). Xenodiagnosis was performed on 65% of them: 36/38 of the treated and 9/34 of the untreated patients had previous positive xenodiagnosis. Post-treatment, 133 xenodiagnoses were performed on 41 patients, all resulting negative. In the control group, 29 xenodiagnoses were performed on 14 patients; 2 resulted positive. Sera stored during the follow-up were simultaneously analyzed through conventional serology tests (IHA; DA-2ME; IIF). The serological evolution in the treated group was: a) 37% underwent negative seroconversion (nifurtimox 11, benznidazole 9); b) 27.8% decreased titers (nifurtimox 9, benznidazole 6), 9 showed inconclusive final serology (nifurtimox 7, benznidazole 2); c) 35.2% remained positive with constant titers (nifurtimox 7; benznidazole 12). The control group conserved the initial antibody levels during the follow-up. In the clinical evolution, 2/54 (3.7%) of the treated and 9/57 (15.8%) of the untreated patients showed electrocardiographic disturbances attributable to Chagas myocardiopathy, with a statistically relevant difference (p<0.05). Treatment caused deparasitation in at least 37% of the chronically infected adults and a protective effect on their clinical evolution.

Deterioração bacteriológica do jaraqui Semaprochilodus spp. capturado no estado do Amazonas e conservado em gelo

Neste trabalho foram analisados 73 espécimes de jaraqui Semaprochilodus spp. conservados em caixas de poliestireno expandido entre camadas de gelo. Foram realizadas as seguintes análises: avaliação sensorial pela tabela de Torry modificada e pelo índice de qualidade por deméritos; determinação do pH e das bases voláteis totais (N-BVT); contagem total dos microrganismos aeróbios psicrófilos a 20 ºC por 4 dias, psicrotróficos a 7 ºC por 10 dias, dos mesófilos a 37 ºC por 2 dias; contagem, isolamento e identificação das bactérias Aeromonas sp. Bacillus sp. e Pseudomonas sp. a 20 ºC por 24 horas e de Plesiomonas sp. a 37 ºC por 2 dias. O jaraqui se manteve em condições de consumo, pela avaliação sensorial, por 18 e 21 dias. O pH e as bases voláteis totais não foram bons indicadores de qualidade; as contagens totais de psicrófilos, psicrotróficos e mesófilos não apresentaram diferença significativa e as bactérias não apresentaram comportamento deteriorador pela ausência da produção de H2S.

Aproveitamento do Óleo das Amêndoas de Tucumã do Amazonas na Produção de Biodiesel

A falta de disponibilidade de energia elétrica é um dos principais motivos pelo baixo Índice de Desenvolvimento Humano das comunidades isoladas localizadas na Amazônia. O biodiesel produzido a partir de óleos vegetais extraídos de espécies oleaginosas nativas de forma sustentada é uma das melhores alternativas energéticas para a região. O tucumã do amazonas, Astrocaryum aculeatum, é uma espécie de palmeira que produz um fruto muito apreciado na região, a partir do qual se obtém uma amêndoa com alto teor de óleo. Nesse estudo, foi avaliada a produção de biodiesel etílico, a partir de diferentes lotes de óleos de tucumã do amazonas, com índices de acidez baixos e elevados, pela transesterificação por catálise básica e ácida homogêneas, respectivamente. Na catálise ácida, foram testados HCl e H2SO4 como catalisadores nas concentrações de 0,0625 a 1,000 M, empregando etanol hidratado na proporção molar de 1:6 e a reação conduzida a 90 ºC por 24 h. Na catálise básica, foram testados NaOH e KOH, nas proporções de 0,5 a 2,0 %, empregando etanol anidro na proporção molar de 1:12 e a reação conduzida a 80 ºC por 2 h. O biodiesel obtido em cada experimento foi analisado por métodos físicos (massa específica) e cromatográficos (CLAE em fase reversa). Análises cromatográficas indicaram que as melhores conversões foram alcançadas por amostras de biodiesel com massas específicas inferiores a 0,87 g.cm-1. As amostras de biodiesel obtidas com melhor qualidade foram obtidas utilizando-se os catalisadores ácidos a 1,0 M com rendimentos superiores a 90%. No caso da catálise básica, obteve-se biodiesel de boa qualidade empregando-se o catalisador NaOH a 2,0%, porém com rendimento inferior a 60 %. Contudo, em ambos os casos, foi possível identificar um excelente potencial de produção de biocombustível, a partir do óleo das amêndoas de tucumã.

Comparação do perfil lipídico, pressão arterial e aspectos nutricionais em adolescentes, filhos de hipertensos e de normotensos

OBJETIVO: Comparar a pressão arterial, o perfil lipídico, o consumo alimentar e dados antropométricos em adolescentes com ou sem antecedente familiar de hipertensão arterial. MÉTODOS: Foram avaliados 43 adolescentes de ambos os sexos, na faixa etária entre 11 a 18 anos, sendo 20 filhos de hipertensos e 23 de normotensos e examinados: a pressão arterial, o consumo alimentar, dados antropométricos, o perfil lipídico e o resultado da orientação dietética (American Heart Association). RESULTADOS: Os filhos dos hipertensos mostraram maiores valores basais de pressão arterial sistólica (109 ± 3 vs. 99 ± 2 mm Hg, p=0,01) e diastólica (68 ± 2 vs. 62 ± 2 mm Hg, p=0,04), da relação CT/HDL-c (4,1 ± 0,3 vs. 3,2 ± 0,2, p<0.01) e de LDL-c/HDL-c (2,7 ± 0,2 vs. 1,9 ± 0,1, p<0,01) e menores valores de HDL-c (43 ± 2 vs. 53 ± 2 mg/dL, p<0,005). O consumo alimentar e medidas antropométricas analisadas não diferiram entre os grupos. A intervenção dietética, embora tenha resultado em reduções no índice de massa corpórea (21,0± 1,2 vs. 20,1 ± 1,1 kg/m², p<0,01), não modificou a dislipidemia presente nos filhos de hipertensos. CONCLUSÃO: Encontraram-se maiores níveis de pressão arterial e perfil lipídico mais desfavorável entre filhos de hipertensos, onde os níveis baixos de HDL-c foram o achado mais relevante e independente de variáveis antropométricas ou nutricionais.