Estudio del efecto fisiológico del consumo de arepas enriquecidas con pectina extraída de la cáscara de curuba (Passiflora tripartita var. mollissima) / Study of the physiological effect of the consumption of arepas enriched with pectin extracted from banana passionfruit peel (Passiflora tripartita var. mollissima) / Estudo do efeito fisiológico pelo consumo de arepas enriquecidas com pectina extraída da casca de curuba (Passiflora tripartita var. mollisima)

Resumen Se evaluó el efecto fisiológico del consumo de arepas de maíz enriquecidas con 10% (p/p) de pectina extraída de cáscara de curuba sobre los niveles séricos de glucosa y lípidos en adultos voluntarios. La pectina se extrajo con ácido clorhídrico y se precipitó con etanol del 96% (v/v). En primer lugar, los voluntarios consumieron arepa sin pectina por siete días; después, arepa con 10% (p/p) de pectina cítrica comercial por 20 días y, finalmente, arepa con 10% de pectina de curuba por 20 días. Al mismo tiempo, se realizó un análisis sensorial de aceptación. Los resultados mostraron que, al consumir arepa con pectina de curuba, los niveles séricos de glucosa disminuyeron significativamente (5,88% (p = 0,023)), mientras que el perfil lipídico no se afectó. La arepa con pectina de curuba mostró mayor porcentaje de fibra dietética soluble, insoluble y total, así como menor porcentaje de carbohidratos disponibles, respecto a las otras arepas analizadas. El panel sensorial dictaminó baja aceptación en sabor y textura para las arepas con pectina cítrica comercial y con pectina de curuba. Se concluyó que la pectina de curuba puede aprovecharse para la formulación de arepas ya que ofrece a los consumidores un alimento benéfico para reducir los niveles de glucosa en la sangre.

Abstract The physiological effect of corn arepas consumption enriched with 10% (w/w) pectin extracted from banana passionfruit peel was evaluated on the serum levels of glucose and lipid in adult volunteers. The pectin was extracted with hydrochloric acid and precipitated with 96% (v/v) ethanol. Firstly, the volunteers consumed arepa without pectin for seven days; then, arepa with 10% of commercial citrus pectin for 20 days, and, finally, arepa with 10% banana passionfruit pectin for 20 days. At the same time, a sensory acceptance analysis was carried out. The results showed that, when consuming arepa with banana passionfruit pectin, serum glucose levels decreased significantly (5.88% (p = 0.023)), while the lipid profile was not affected. The arepa formulated with banana passionfruit pectin showed a higher percentage of soluble, insoluble and total dietary fiber, as well as a lower percentage of available carbohydrates as compared to the other analyzed arepas. The sensory panel ruled low acceptance in flavor and texture for arepas with commercial citrus and banana passionfruit pectin. It was concluded that banana passionfruit pectin can be used for the formulation of arepas since it offers to consumers a beneficial food to reduce blood glucose levels.

Resumo Este estudo avaliou o efeito metabólico do consumo de arepas de milho enriquecidas com 10% (p/p) de pectina extraída da casca da curuba, sobre os níveis séricos de glucose e lipídios, em adultos voluntários. A pectina foi extraída utilizando ácido clorídrico e precipitou-se com etanol à 96% (v/v). Primeiro, os voluntários consumiram arepa sem pectina por sete dias, então arepa com 10% de pectina cítrica comercial por 20 dias, e finalmente arepa com 10% de pectina de curuba por 20 dias. Paralelamente, foi realizado uma análise sensorial de aceitação. Os resultados encontrados mostram que ao consumir arepa com pectina de curuba os níveis séricos de glucose diminuíram significativamente (5,88% (p = 0,023)), enquanto que o perfil lipídico não foi afectado. A arepa com pectina de curuba mostrou maior percentagem de fibra dietética solúvel, insolúvel e total, e um menor percentagem de carboidratos disponíveis com respeito aos valores das outras arepas avaliadas. O painel sensorial mostrou baixa aceitação no sabor y textura para arepas com pectina cítrica comercial e de curuba. Concluiu-se que a pectina de curuba pode ser usada para a formulação de arepas, uma vez que oferece aos consumidores um alimento benéfico para reduzir os níveis de glucose no sangue.

Dynamics of the T cell response to Mycobacterium tuberculosis DosR and Rpf antigens in a Colombian population of household contacts of recently diagnosed pulmonary tuberculosis patients.

Immune response to DosR and Rpf antigens from Mycobacterium tuberculosis (Mtb) seems to be important for latency maintenance. Little is known about the dynamics of the immune response to these antigens in an endemic community. Thus, the IFNγ response and cytokine production in response to PPD, Esat6-Cfp10 (E6-C10), DosR and Rpf antigens in healthy HHC of tuberculosis (TB) patients over a 12 (T12) months period (short-term, stLTBI) was investigated. This response was compared with a group of LTBI, who have remained healthy for 5-7 years (long-term, ltLTBI). According to the IFNγ response, two groups of HHCs were identified in stLTBI in response to E6-C10. At T12, E6-C10(+) HHCs displayed a decrease in the IFNγ levels and a generalized decrease in cytokines production. The E6-C10(-) HHC showed an increase in the IFNγ response and cytokine levels. In stLTBI, the responses to E6-C10, DosR, and Rpf may be interpreted as a protective immune response controlling Mtb infection and may be leading to a state of latent infection. Comparing the response of stLTBI and ltLTBI, we observed significant changes in the proportions of CD45RO(+)CD27(+) T cells to specific DosR and Rpf, which may indicate a persistent immune response to Mtb antigens in ltLTBI.

Increased percentage of IFN-γ producing CD56+CD3+ cells in active tuberculosis patients upon CFP-10 stimulation of peripheral mononuclear cells.

Aiming to identify a possible biomarker that distinguishes immune cellular response of active tuberculosis from latent infection. Peripheral blood mononuclear cells (PBMCs) of pulmonary tuberculosis patients (PTB), tuberculin positive household contacts (TST(+) HHC), and tuberculin negative non-household contacts (TST− Non HHC) were stimulated with PPD or CFP-10 and the percentage of CD69(+) cells, proliferating precursor and IFN-γ producing CD4(+), CD8(+), CD56(+)CD3(−) and CD56(+)CD3(+) cells were compared. IL-2, IL-12p70, IL-15, IL-18 and IL-10 were measured in culture supernatants. PTB and TST+ HHC presented higher percentages of CD69(+) cells, IFN-γ(+) and proliferating precursors in all subpopulations studied and higher IL-12p70 levels than TST- Non HHC. The increased percentage of IFN-γ producing CD56(+)CD3(+) cells in response to CFP-10 in PTB, compared with TST− Non HHC and the ratios between the percentage of CD56(+)CD3(+) cells/CD56(+)CD3(−) and CD8(+) cells producing IFN-γ suggest that these parameters may distinguish active TB from latently infected individuals.

Contenido total de fenoles y actividad antioxidante de pulpa de seis frutas tropicales / Total phenolic content and antioxidant activity of pulp extracts of six tropical fruits / Conteúdo total de fenóis e atividade antioxidante em alguns trechos da polpa de frutas tropicais

Seis pulpas de frutas tropicales de origen colombiano: curuba (Passiflora tripartida var. Mollissima), gulupa (Passiflora edulis Sims), aguacate variedad Hass (Persea America Mill), lulo (Solanum quitoense Lam.), tomate de árbol (Cyphomandra betacea Sendt) y uchuva (Physalis peruvianaL) fueron empleadas para realizar el estudio del contenido total de fenoles, por el método de Folin Ciocalteu y la actividad antioxidante por los métodos químicos: DPPH (2,2-difenil-2-picrilhidrazilo) y FRAP (poder antioxidante para reducir iones férricos), y por los métodos biológicos: oxidación de lipoproteínas de baja densidad (LDL) e inhibición del estrés oxidativo sobre el crecimiento de Saccharomyces cerevisiae. Se encontró que el extracto de curuba presentó el mayor contenido total de fenoles con un valor de 683,48 ± 18,48 mg equivalentes de ácido gálico/100 g muestra B.H. (Base Húmeda). En cuanto a los ensayos de actividad antioxidante el extracto de aguacate presentó mayor actividad por el método DPPH con un valor de 165,10 ± 4,36 µmol Trolox/100 g muestra B.H., mientras que el extracto de curuba presentó la mayor actividad antioxidante por el método FRAP con un valor de 148,07 ± 12,07 µmol Trolox/g muestra B.H. En el ensayo de oxidación de LDL los extractos de curuba y uchuva presentaron la mayor capacidad de inhibición en la oxidación de dichas lipoproteínas con un valor de 1,61 ± 0,01 y 1,46 ± 0,06 nmol TMP (Tetrametoxipropano)/g muestra B.H., respectivamente. En el ensayo de inhibición del estrés oxidativo sobre el crecimiento de S. cerevisiae los extractos de aguacate, curuba y gulupa tuvieron el mayor efecto protector obteniéndose una concentración celular de.

Six pulp extracts of tropical fruits from Colombia: banana passion fruit (Passiflora tripartida var. Mollissima), gulupa (Passiflora edulis Sims), avocado (Persea americana Mill.), lulo (Solanum quitoense Lam.), tomate de árbol (Cyphomandra betacea Sendt.), and gooseberry (Physalis peruviana L.) were used for studying the total phenolic content by the Folin Ciocalteu method and the antioxidant activity by the chemical methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) and FRAP (ferric ion reducing antioxidant power), and by the biological methods: oxidation of low density lipoproteins (LDL) and inhibition of oxidative stress on Saccharomyces cerevissiae growth. It was found that banana passion fruit extract showed the highest content of total phenolics, with a value of 683.48 ± 18.48 mg equivalents to gallic acid/100 g fresh weight (F.W.). Regarding the antioxidant activity the avocado extract exhibited the highest activity in the DPPH assay, with a value of 165.10 ± 4.36 µmol Trolox/100g F.W. whereas the banana passion fruit extract showed the highest antioxidant activity by the FRAP method, with a value of 148.07 ± 12.07 µmol Trolox/g F.W. In the LDL oxidation assay the extracts from banana passion fruit and gooseberry presented the greater ability to inhibit the oxidation of those lipoproteins, with a value of 1.61 ± 0.01 and 1.46 ± 0.06 nmol TMP (Tetrametoxipropane)/g F.W., respectively. In the assay of inhibition of oxidative stress on S. cerevissiae the avocado, banana passion fruit, and gulupa extracts exhibited the main protective effect and the obtained values for cellular concentration were 1.96 ×10(7) cells/mL, 1.86 × 10(7) cells/mL, and 1.54 × 10(7) cells/mL, respectively, as compared to 0.99 × 10(7) cells/mL found.

Seis polpa de frutas tropicais da Colômbia: maracujá (Passiflora tripartite Mollissima var.), Gulupa (Passiflora edulis Sims), Hass abacate (Persea América Mill), lulo (Solanum quitoense Lam.), tomate de árvore (Cyphomandra betacea Sendt) e uchuva (Physalis Peruviana L) foram utilizados para o estudo do teor total de fenol pelo método de Folin-Ciocalteu e actividade antioxidante por métodos químicos: DPPH (2,2-difenil-2-picrilhidrazilo) e FRAP (poder antioxidante de reduzir iões férricos), e métodos biológicos: a oxidação de lipoproteínas de baixa densidade (LDL) e inibição de estresse oxidativo no crescimento de Saccharomyces cerevisiae. Os resultados são encontrados no extrato tiveram o maior teor total maracujá de fenóis com um valor de 683,48 ± 18,48 mg equivalentes o ácvido gálico/100 g amostra de ácido BH (Base úmida). Quanto à atividade antioxidante testes abacate extrato apresentou maior atividade com o método DPPH vale 165,10 ± 4,36 µmol Trolox / 100g de amostra BH, enquanto o extrato de maracujá apresentou a maior atividade antioxidante, método FRAP com um valor de 148,07 ± 12,07 µmol Trolox/g amostra BH. No ensaio de oxidação de LDL e extrai maracujá e uchuva teve a maior capacidade para inibir a oxidação de lipoproteínas estes com um valor de 1,61 ± 0,01 e 1,46 ± 0,06 nmol TMP (tetrametoxipropano)/g BH amostra, respectivamente. No ensaio de inibição de estresse oxidativo no crescimento de S. cerevisiae de extratos de abacate, maracujá e gulupa tinha o maior efeito protector sendo obtido uma concentração de células de 1,96 ×10(7) células/mL, 1,86 × 10(7) células/ml e 1,54 × 10(7) células/mL, respectivamente, em comparação com aqueles encontrados.

Estudio comparativo del efecto metabólico de arepas enriquecidas con pectina extraída de guayaba (Psidium guajava L.) o pectina cítrica comercial / Comparative study of the metabolic effect of arepas enriched with pectin extracted from guava (Psidium guajava L.) or commercial citrus pectin / Estudo comparativo do efeito metabólico de arepas enriquecidas com pectina extraída de goiaba (Psidium guajava L.) ou pectina cítrica comercial

La guayaba (Psidium guajava L.), contiene hasta un 8.5% de fibra dietética total de la cual el 1.9% es fibra soluble, lo que hace a esta fruta tropical una buena fuente de pectina, fibra soluble cuyo consumo disminuye la absorción intestinal de los lípidos y protege contra las enfermedades cardiovasculares. En este trabajo se evaluó el efecto metabólico del consumo de arepas de maíz enriquecidas con un 10% de pectina durante 20 días, en el perfil lipídico y la glicemia de un grupo de 75 estudiantes universitarios; se buscó además establecer las diferencias en el efecto observado cuando dentro de la formulación de las arepas se adicionó una pectina cítrica de uso comercial respecto a cuando se empleó pectina extraída de guayaba (Psidium guajava L.). La comparación de los niveles de glucosa y lípidos antes y después del tratamiento mostró que el consumo de dichas arepas generó cambios favorables para la salud de los individuos, pues hubo una disminución estadísticamente significativa en los niveles séricos de colesterol total, colesterol LDL y glucosa. Aunque hay una tendencia en los resultados que muestra un aumento promedio en el colesterol HDL luego del consumo de los productos con los dos tipos de pectina, no hay evidencia estadística para inferir que dicho cambio sea significativo. De la misma forma, los resultados no indican diferencias entre el efecto generado por lo dos tipos de pectina, a excepción de la mayor disminución en los niveles de triglicéridos lograda con la adición de pectina comercial.

Guava (Psidium guajava L.), contains up to 8.5% total dietary fiber, of which 1.9% is soluble fiber, which makes this tropical fruit a good source of pectin, soluble fiber whose consumption decreases intestinal absorption of lipids and protects against cardiovascular disease. In this work the metabolic effect of consumption of corn arepas enriched with 10% pectin for 20 days, in the lipid profile and blood glucose in a group of 75 university students was evaluated; it was also seek to establish the differences in the effect observed when within the formulation of the arepas a commercial citrus pectin was added compared to when pectin extracted from guava (Psidium guajava L.) was used. The comparison of the glucose and lipid levels before and after treatment showed that with consumption of those arepas favorable changes for the health of the individuals were generated, since there was a statistically significant decrease in serum levels of total cholesterol, LDL cholesterol and glucose. Although there is a trend in the results showing an average increase in HDL cholesterol after consumption of products with the two types of pectin, there is no statistical evidence to infer that the change is significant. Similarly, the results indicate no difference between the effects generated by the two types of pectin, except for the greatest decrease in triglyceride levels achieved when commercial pectin was added.

A goiaba (Psidium guajava L.) contém até 8,5% de fibra alimentar total, dos quais 1,9% é fibra solúvel, o que torna esta fruta tropical uma boa fonte de pectina, fibra solúvel, cujo consumo diminui a absorção intestinal de lipídios e protege contra doenças cardiovasculares. Este estudo avaliou o efeito metabólico do consumo de arepas de milho enriquecidas com 10% de pectina, durante 20 dias, no perfil lipídico e glicêmico de um grupo de 75 estudantes universitários. Também se procurou determinar as diferenças no efeito observado quando na formulação das arepas se adicionou uma pectina cítrica comercial comparado com o uso de uma pectina extraída de goiaba (Psidium guajava L.). A comparação dos níveis de glicose e lípidos, antes e depois do tratamento, mostrou que o consumo destas arepas originou mudanças favoráveis para a saúde dos indivíduos, uma vez que houve uma diminuição estatisticamente significativa nos níveis séricos do colesterol total, colesterol LDL e glicose. Embora haja uma tendência nos resultados que mostra um aumento médio nos níveis de colesterol HDL após o consumo de produtos com os dois tipos de pectinas, não há nenhuma evidência estatística para inferir que a alteração seja significativa. Da mesma forma, os resultados indicam que não há diferença entre o efeito gerado pelos dois tipos de pectina, exceto para a maior diminuição nos níveis de triglicerídeos obtidos com a adição de pectina comercial.

Stable extracellular RNA fragments of Mycobacterium tuberculosis induce early apoptosis in human monocytes via a caspase-8 dependent mechanism.

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis.

Análisis proteóiico de cultivos primarios de tiroides / Proteomic nalysiis of primary cultures from thyroid / Análiseproteómicco de culturas preliminares do thyroid

En el presente estudio se identificaron proteínas de expresión constitutiva, como vimentina, actina, tubulina, proteína de choque térmico de 60 kDa, peroxirredoxina y la ATP sintasa mitocondrial, en cultivos primarios de tiroides normales y de carcinoma papilar de tiroides. Se establecieron las condiciones de extracción, solubilización, análisis cuantitativo y cualitativo de dichas proteínas, y se buscaron las mejores condiciones de isoelectroenfoque (IEF) en la electroforesis en dos dimensiones (2D). En la extracción y solubilización de las proteínas se evaluó la presencia o ausencia de anfolitos y sales, se obtuvo un mejor resultado empleando en el amortiguador de extracción sales como Tris-HCl y acetato de magnesio que incrementan la solubilidad de las proteínas. Para la cuantificación se recomienda el uso conjunto de técnicas colorimétricas con la electroforesis SDS-PAGE tiñendo con azul de Coomassie y corroborando los resultados mediante western blot, lo cual permite, además, verificar la integridad de las proteínas. Respecto a la electroforesis en dos dimensiones, se obtuvieron geles con un mayor número de manchas (spots),resueltos, enfocados y reproducibles empleando en el IEF gradientes inmovilizados de pH de 4-7 y voltaje final de 8.000 V. Las proteínas se identificaron mediante el análisis bioinformático de los geles 2D con el programa PDQuest (PDQuest 7.2, Bio-Rad®) y MALDI-TOF.

In this paper, proteins of constitutive expression such as vimentin, actin, tubulin, heat shock protein of 60 kDa, peroxiredoxin and the mitochondrial ATP sintase were identified in primary cultures of normal thyroid and papillary carcinoma. The extraction conditions, solubilization, quantitative and qualitative analysis of such proteins were established. In addition, the best conditions for isoelectrofocusing (IEF) in the two-dimensional electrophoresis (2D) were founded. In the extraction and solubilization of proteins, the presence or absence ofampholytes or salts was evaluated. The best result in the extraction was obtained using buffer salts such as Tris-HCl and magnesium acetate, which increase the solubility of these proteins. For quantification, the recommendation is to combine colorimetric techniques with SDS-PAGE electrophoresis stained with Coomasie blue and Western blot to confirm the results, which allows verifying the integrity of these proteins. In the two-dimensional electrophoresis, resolved, focused and reproducible gels with greater number of spots were obtained using in the IEF immobilized pH gradients of 4-7 and final voltage of 8000 V. The proteins were identified by the bioinformatic analysis of the 2DE gels with the PDQuest program (PDQuest 7.2 (BioRad®) and MALDI-TOF.

Neste estudo, se identificaram proteínas da expressão constitutiva, como vimentina, actina, tubulina, proteína de choque do calor do kDa 60, peroxiredoxinaeasintase mitocondrial do ATP, em cultivos primários de tireóide normal e de carcinoma papilar de tireóide. Se estabeleceram as condições de extração e de solubilization, assim como a análise quantitativaeaqualitativa de tais proteínas, e procuraram-se asmelhores condições para isoeletroenfoque (IEF) na eletroforese bidimensional (2D). Na extração e no solubilization das proteínas, se avaliou a presença ou a ausência de anfolitos ou de sais. O melhor resultado foi obtido usando sais como Tris-HCl e acetato do magnésio no amortecedor da extração, as quais aumentam a solubilida-de das proteínas. Na quantificação, se recomenda o uso de técnicas colorimétricas com eletroforese SDS-PAGE, tingindo com azul de Coomasie. Os resultados se confirmam com Western blot, o qual permite também verificar a integridade das proteínas. Quanto à eletroforese bidimensional, se obtiveram géis com um maior número de pontos (spots) determinados, enfocados e reprodutíveis no IEF usando gradientes imobilizados de pH 4-7 e tensão final de 8000 V. As proteínas foram identificadas por análise bioinformática de géis 2D com o programa PDQuest (PDQuest 7.2 (Bio-Rad®) e MALDITOF.

IFNgamma response to Mycobacterium tuberculosis, risk of infection and disease in household contacts of tuberculosis patients in Colombia.

OBJECTIVES: Household contacts (HHCs) of pulmonary tuberculosis patients are at high risk of Mycobacterium tuberculosis infection and early disease development. Identification of individuals at risk of tuberculosis disease is a desirable goal for tuberculosis control. Interferon-gamma release assays (IGRAs) using specific M. tuberculosis antigens provide an alternative to tuberculin skin testing (TST) for infection detection. Additionally, the levels of IFNgamma produced in response to these antigens may have prognostic value. We estimated the prevalence of M. tuberculosis infection by IGRA and TST in HHCs and their source population (SP), and assessed whether IFNgamma levels in HHCs correlate with tuberculosis development. METHODS: A cohort of 2060 HHCs was followed for 2-3 years after exposure to a tuberculosis case. Besides TST, IFNgamma responses to mycobacterial antigens: CFP, CFP-10, HspX and Ag85A were assessed in 7-days whole blood cultures and compared to 766 individuals from the SP in Medellín, Colombia. Isoniazid prophylaxis was not offered to child contacts because Colombian tuberculosis regulations consider it only in children under 5 years, TST positive without BCG vaccination. RESULTS: Using TST 65.9% of HHCs and 42.7% subjects from the SP were positive (OR 2.60, p<0.0001). IFNgamma response to CFP-10, a biomarker of M. tuberculosis infection, tested positive in 66.3% HHCs and 24.3% from the SP (OR = 6.07, p<0.0001). Tuberculosis incidence rate was 7.0/1000 person years. Children <5 years accounted for 21.6% of incident cases. No significant difference was found between positive and negative IFNgamma responders to CFP-10 (HR 1.82 95% CI 0.79-4.20 p = 0.16). However, a significant trend for tuberculosis development amongst high HHC IFNgamma producers was observed (trend Log rank p = 0.007). DISCUSSION: CFP-10-induced IFNgamma production is useful to establish tuberculosis infection prevalence amongst HHC and identify those at highest risk of disease. The high tuberculosis incidence amongst children supports administration of chemoprophylaxis to child contacts regardless of BCG vaccination.

LA ACTIVACIÓN DE LA VÍA DE SEÑALIZACIÓN PI3K/AKT POR EL FACTOR DE CRECIMIENTO SIMILAR A LA INSULINA TIPO II ESTIMULA LA EXPRESIÓN DEL mARN DE LA METALOPROTEINASA 9 EN CÉLULAS DE CORIOCARCINOMA / ACTIVATION OF THE PI3K/AKT SIGNALLING PATHWAY BY INSULIN-LIKE GROWTH FACTOR TYPE II STIMULATES METALLOPROTEINASE 9mRNA EXPRESSION IN CHORIOCARCINOMA CELLS / PERCURSO SINALIZAÇÃO PI3K/AKT ACTIVADO POR INSULINA-COMO FACTOR DE CRESCIMENTO TIPO II ESTIMULA METALOPROTEINASE 9 mARN EXPRESION CORIOCARCINOMA EM CÉLULAS

Las metaloproteinasas 2 y 9 (MMP-2 y MMP-9) y el inhibidor-1 de MMP (TIMP-1) podrían contribuir a la regulación del comportamiento invasivo de las células trofoblásticas, efecto que podría ser mediado por el factor de crecimiento similar a la insulina tipo II (IGF-II), el cual regula el desarrollo y la función del trofoblasto en la interfase materno-fetal. El objetivo de este estudio fue investigar el rol de la vía de señalización PI3K/AKT activada por IGF-II en la expresión de MMP-2 y MMP-9 involucradas en el proceso de invasión en la línea celular JEG-3, de coriocarcinoma humano. La expresión del mARN de MMP-2, MMP-9 y TIMP-1 fue evaluada, en dichas células, por la técnica de RT-PCR empleando diferentes dosis de IGF-II y diferentes condiciones, mientras que la vía de señalización fue evaluada usando la técnica de Western blot. Se encontró que el IGF-II no contribuye a la proliferación de las células trofoblásticas; sin embargo, este promueve la expresión del mARN de MMP-9 y TIMP-1 pero no de MMP-2, de forma dependiente de la dosis. El efecto en la expresión de MMP-9 es mediado a través de IGF-II por la activación de la vía de señalización PI3K/ AKT después de la fosforilación del receptor de IGF-I (IGF-IR). De acuerdo con los resultados, se propone un modelo en el cual la interacción de IGF-II con IGF-IR conduce a la activación de PI3K/AKT y la subsecuente expresión y activación de MMP-9. Esta activación es un requisito esencial para el proceso invasivo.

Metalloproteinases 2 and 9 (MMP-2 and MMP-9) and TissueInhibitor-1 ofMMPs (TIMP-1) could contribute to regulate the invasive behaviour of trophoblastic cells, effect which could be mediated by Insulin-like growth factor type II (IGF-II), which regulates the development and function of trophoblast at the foetal-maternal interface. The aim of this study was to investigate the role of the PI3K/AKT signalling pathway activated by IGF-II in the expression of MMP-2 and MMP-9 involved in the invasion process in the human choriocarcinoma cell line JEG-3. MMP-2, MMP-9 and TIMP-1 mARN expression in those cells were evaluated by RT-PCR using different IGF-II doses and conditions while signalling pathway was evaluated using Western blot. It was found that IGF-II does not contribute to trophoblastic cells proliferation, however, it promotes mARN expression of MMP-9 and TIMP-1, but not of MMP-2, in a dose-dependent way. The effect on MMP-9 expression is mediated through IGF-II activation of PI3K/AKT signalling pathway after phosphorylation of the IGF-I receptor (IGF-IR). According with the results a model is proposed in which the interaction of IGF-II with IGF-IR leads to PI3K/AKT activation and subsequent expression and activation of MMP-9. This activation is an essential requirement for the invasive process.

As metaloproteinasas 2 y 9 (MMP-2 y MMP-9) e o inibidor-1 de MMPs (TIMP-1) poderiam contribuir à regulação do comportamento invasivo das células trofoblásticas, efeito que poderia ser mediado pelo Factor de Crescimento Similar à Insulina tipo II (IGF-II), o qual regula o desenvolvimento e função do trofoblasto na interface materno-fetal. O objectivo deste estudo foi investigar o papel da via de sinalização PI3K/AKT activada por IGF-II na expressão de MMP-2 e MMP-9 envolvidos no processo de invasão na linha celular JEG-3, de coriocarcinoma humano. A expressão do mARN de MMP-2, MMP-9 y TIMP-1, foi avaliada, em ditas células, pela técnica de RT-PCR utilizando diferentes doses de IGF-II e diferentes condições, enquanto que a via de sinalização foi avaliada usando a técnica de Western-blot. Foi encontrado que o IGF-II não contribui à proliferação das células trofoblásticas, no entanto, este promoveu a expressão do mARN de MMP-9 e TIMP-1 mas não de MMP-2, de forma dependente da dose. O efeito na expressão de MMP-9 é mediado através de IGF-II pela activação da via de sinalização PI3K/AKT depois da fosforilação do receptor de IGF-I (IGF-IR). De acordo aos resultados é proposto um modelo, no qual a interacção de IGF-II com IGF-IR conduz à activação de PI3K/AKT e à subsequente expressão e activação de MMP-9. Esta activação é um requisito essencial para o processo invasivo.

RELACIÓN DE LA EXPRESIÓN DE mARN DE LAS METALOPROTEINASAS 2 Y 9 Y EL INHIBIDOR TIMP-1 EN MOLA HIDATIDIFORME COMPLETA / RELATIONSHIP BETWEEN METALLOPROTEINASES 2 AND 9 AND TIMP-1 INHIBITOR mRNA EXPRESSION IN HYDATIDIFORME MOLE / RELAÇÃO DA EXPRESSÃO DE mRNA DAS METALOPROTEINASAS 2 E 9 E DO INHIBIDOR TIMP-1 NA MOLA HIDATIDIFORME COMPLETA

Durante el comienzo de la gestación el factor de crecimiento similar a la insulina tipo II (IGF-II) se expresa abundantemente en la placenta y podría regular el comportamiento invasivo de las células trofoblásticas. La habilidad invasiva dependerá también de la capacidad de secretar metaloproteinasas (MMPs), cuya expresión anormal contribuye a varios procesos patológicos. En la Enfermedad Trofoblástica Gestacional (ETG) estos factores podrían estar desregulados, así la mola hidatidiforme completa (MHC) conduce a la formación del coriocarcinoma, que es un tumor invasivo y metastásico. El objetivo de este estudio fue investigar la relación entre los niveles de mARN de IGF-II, MMP-2, MMP-9 y TIMP-1, por la técnica de RT-PCR, en tejidos de mola hidatidiforme vs. placentas de primer trimestre. La actividad de las MMPs se evaluó usando el ensayo de zimografía, encontrando que en MHC ésta se incrementa, lo cual podría relacionar la malignización del trofoblasto con el incremento en su capacidad invasiva. La expresión elevada de IGF-II en esta patología, también podría estar asociada con el incremento en la actividad de estas MMPs. Una mayor relación entre la expresión de mARNde MMP-9/TIMP-1 en la mola hidatidiforme completa se sugiere como predictor de malignización del tejido trofoblástico .

At the beginning of gestation the Insulin Like Growth Factor II (IGF-II) is highly expressed in placenta and it could regulate the invasive behavior of trophoblastic cells. This invasive ability depends on its capability to secret metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs), abnormal expression of which could contribute to several pathological processes.In the Gestational Trophoblastic Diseases (GTD) these factors could be unregulated, in this way complete hydatidiforme mole (CHM) produces the choriocarcinoma which is an invasive and metastasic tumor. The aim of this study was to investigate the relationship between IGF-II, MMP-2, MMP-9 and TIMP-1 mRNA levels by RT-PCR technique, in hydatidiform mole tissues vs. first normal trimester placentas. MMPs activity was evaluated using zimography assay finding that in CHM this is increased, which could relate trophoblast malignization with an increased invasive capability. The higher IGF-II mRNA expression in this pathology could, also, be associated with increased MMPs activity. A higher MMP-9/ TIMP-1 mRNA expression ratio in CHM is suggested as a predictor of trophoblastic tissue malignization .

No início da gestação o fator de crescimento semelhante à insulina tipo II (IGF-II) é altamente expresso na placenta e poderia regular o comportamento invasivo das células trofoblásticas. Esta habilidade invasiva também depende da sua capacidade de produzir metaloproteinasas (MMP) e inibidores das mesmas (TIMP), expressão anormal da qual poderia contribuir em vários processos patológicos. Nas Doenças Trofoblásticas da Gestação (Gestational Trophoblastic Disease, ETG) estes fatores poderiam ser desregulados; deste modo a Mola Hidatidiforme Completa (MHC) produz coriocarcinoma que é um tumor invasivo e metastásico. O objetivo deste estudo era pesquisar a relação entre os níveis de mRNA do IGF-II, MMP-2, MMP-9 e TIMP-1, através da técnica de RT-PCR em tecidoos da MHC vs. placenta do primeiro semestre. Atividade das MMP foram avaliadas pelo ensaio de zimografia achando que em MHC aumenta; isto poderia relacionar o incremento do malignidade do trofoblasto com uma capacidade invasiva aumentada. A expressão mais alta do IGF-II nesta patologia pode também estar associada com atividade aumentada destas MMP. Uma relação aumentada da expressão do mRNA de MMP-9/TIMP-1 na MHC é sugerida como um preditor do incremento da malignidade do tecido trofoblástico .

Production of pro-inflammatory cytokines during the early stages of experimental Paracoccidioides brasiliensis infection.

Pro-inflammatory cytokines play an important role in both recruitment and activation of leukocytes migrating into tissues in response to invading pathogens. In this study the production of pro-inflammatory cytokines, determined by ELISA assays, and the recruitment of leukocytes into the lungs of BALB/c mice infected with Paracoccidioides brasiliensis conidia were evaluated during the early stages of infection. The results showed that infected mice had a significant increase in leukocytes in the lung during the first 4 days with a peak at day 2 post-challenge; infiltrates were composed mainly of polymorphonuclear neutrophils (PMN). Pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-alpha), interleukin (IL) 6, IL-1beta and macrophage inflammatory protein (MIP) 2 were produced at elevated levels during the first 4 days post-challenge, but only in pulmonary samples and not in sera. Additionally, during the early stages of infection, overall weight loss was recorded in infected mice. These results suggest that pro-inflammatory cytokines could be responsible for the recruitment of leukocytes into the lung during the early stages of P. brasiliensis infection. In addition, both pro-inflammatory cytokine production and leukocyte recruitment may participate in the control of infection by influencing the organization of the immune response in the host exposed to P. brasiliensis conidia.

Obtención de una sonda específica para evaluar la transcripción del gen STAT5b / Obtaining a specific probe to evaluate STAT5b gene transcription

Se describe la estrategia empleada para clonar, en un vector de expresión, un fragmento de 306 pb del gen de STAT5b de rata, amplificado por la técnica de RT-PCR. Se amplificó una región en el terminal 3' donde se encuentra la mayor diferencia entre las secuencias del cADN de las isoformas STAT5a y STAT5b de rata. La clonación se logró utilizando el nuevo sistema TOPO-TA recomendado para productos de difícil clonación. El fragmento se subclonó en el vector pBlueScrip II SK y se caracterizó mediante secuenciación y análisis de restricción. La subclonación se realizó con el propósito de poder obtener sondas sentido y antisentido de ARN por transcripción in vitro. Esta sonda específica se empleó para cuantificar niveles de mARN de STAT5b en hígado de ratas macho mediante la técnica de hibridización en solución o ensayo de proteción de ARNasas

Obtención de una sonda para cuantificar el mARN del factor activador de la transcripción STAT5 en rata / A new probe for use in quantifying STAT5 transcription activator factor in rat mRNA

Se describe la estrategia empleada para clonar, en un vector de expresión, un fragmento del gen de STAT5 amplificado por la técnica de PCR. Variando la concentración de magnesio y la temperatura de alineación, se logró amplificar el fragmento deseado a partir de un cADN de sTAT5b de ratón. El producto de PCR se clonó y se comprobó la identidad de la sonda mediante análisis de restricción y secuenciación. Esta sonda se usó en el ensayo de protección con ribonucleasa A o hibridización en solución, para cuantificar el mARN de STAT5 en hígado y linfocitos de timo de ratas hembras. Se encontró una mayor expresión del mARN de STAT5 en hígado