Assessment of Antimicrobial and Pesticide Residues in Food Products Sourced from Peasant Family Farming in Chile.

The presence of antimicrobial and pesticide residues in products sourced from peasant family farms selected from eight regions of Chile was determined. A total of 204 samples were analyzed from family farm products, including honey, lettuce, tomato, strawberry, raspberry, lamb meat, and cow's milk. Pesticide residues were found in 43 of 107 samples tested for them, but only 4 samples had concentrations that exceeded the maximum residue limits (MRLs) set by Chilean regulations. As for animal products, 2 of 15 cow's milk samples tested positive for permethrin residues; however, these concentrations did not exceed Chilean MRLs. No pesticide residues were detected in honey samples. As for antimicrobial drugs, 4 of 14 lamb meat samples and 11 of 79 cow's milk samples tested positive for different classes of antimicrobial residues, such as tetracyclines, macrolides, aminoglycosides, and ß-lactams. Among these, only three samples exceeded the MRLs for these matrices. Traces of tetracyclines and sulfonamides were detected in 7 of 29 honey samples. These findings show that these chemical contaminants are present in trace concentrations in foodstuffs produced by peasant family farms in Chile; however, most residues did not exceed the regulatory limits. This study is the first assessment of the presence of residues from antimicrobial drugs and pesticides in food products sourced from peasant family farms in Chile. Data about the current state of drug residues in this segment of food products provide a baseline for efforts to close possible gaps in current surveillance schemes.

Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells.

Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-{alpha}.

Estradiol (E(2)) acts on the endothelium to promote vasodilatation through the release of several compounds, including prostanoids, which are products of arachidonic acid metabolism. Among these, prostacyclin (PGI2) and thromboxane A2 (TXA2) exert opposite effects on vascular tone. The role of different estrogen receptors (ERs) in the PGI2/TXA2 balance, however, has not been fully elucidated. Our study sought to uncover whether E(2) enhances basal production of PGI2 or TXA2 in cultured human umbilical vein endothelial cells (HUVECs), to analyze the enzymatic mechanisms involved, and to evaluate the different roles of both types of ERs (ERalpha and ERbeta). HUVECs were exposed to E(2), selective ERalpha (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole, PPT) or ERbeta (diarylpropionitrile, DPN) agonists and antagonists (unspecific: ICI 182 780; specific for ERalpha: methyl-piperidino-pyrazole, MPP). PGI2 and TXA2 production was measured by ELISA. Expression of phospholipases, cyclooxygenases (COX-1 and COX-2), PGI2 synthase (PGIS), and thromboxane synthase (TXAS) was analyzed by western blot and quantitative RT-PCR. E(2) (1-100 nM) dose dependently increased PGI2 production (up to 50%), without affecting TXA2 production. COX-1 and PGIS protein and gene expressions were increased, whereas COX-2, phospholipases, and TXAS expression remained unaltered. All these effects were mediated through ERalpha, since they were produced not only in the presence of E(2), but also in that of PPT, while they were abolished in the presence of MPP. In conclusion, E(2), acting through ERalpha, up-regulates COX-1 and PGIS expression, thus directing prostanoid balance toward increased PGI2 production.

Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells.

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxygenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.

Therapeutic dosages of oral or transdermal estradiol did not modify sCD40L levels in postmenopausal women.

The CD40/CD40L system is considered a crucial modulator of the inflammatory process underlying the progression and complication of atheroma plaques. The soluble fraction of CD40L (sCD40L) is a reliable indicator of the CD40/CD40L system. Our purpose was to investigate whether a therapeutic dose of estradiol, by either the oral or the transdermal route, was associated with changes in circulating levels of sCD40L. Forty-seven women completed a 4-week course of treatment with either oral estradiol valerate (2 mg/day, 20 women) or transdermal estradiol (50 microg/day, 27 women). Serum levels of sCD40L were measured by conventional enzyme-linked immunosorbent assay. Oral, but not transdermal estradiol, modified the lipid profile. Levels of sCD40L, however, remained unchanged compared with baseline. This neutral effect of oral or transdermal estradiol on sCD40L levels further advances our knowledge on the effects of estrogens on mechanisms involved in the progression and complication of atherosclerosis.

Transdermal estradiol reduces F2alpha-isoprostane levels in postmenopausal women.

OBJECTIVE: F2alpha-isoprostanes are considered the most reliable index of in vivo oxidative stress. Given the implication of oxidative stress in the pathogenesis of atherosclerosis, we investigated the effects of hormone therapy on the plasma levels of F2alpha-isoprostanes. DESIGN: Sixty-one healthy postmenopausal women were treated in a randomized trial with estradiol either orally (2 mg/day, 28 women) or transdermally (50 mug/day, 33 women) for 4 weeks. Then women in each group were randomly assigned to oral progestogen, either micronized progesterone (300 mg/day) or medroxyprogesterone acetate (5 mg/day) for 2 additional weeks. Plasma samples were collected before and at the end of each treatment period, either estradiol alone or estradiol plus progestogen. F2alpha-isoprostanes were measured by a commercial enzyme immunoassay. RESULTS: A significant reduction in the levels of F2alpha-isoprostanes was detected only in women receiving transdermal estradiol, alone or in combination with medroxyprogesterone acetate. CONCLUSIONS: Transdermal estradiol alone or associated with medroxyprogesterone acetate decreased plasma levels of F2alpha-isoprostanes. These data elucidate additional details of the beneficial effect of estradiol on oxidative stress, a relevant mechanism in atherogenesis.

Raloxifene increases proliferation of human endothelial cells in association with increased gene expression of cyclins A and B1.

OBJECTIVE: To examine the proliferative effect of of raloxifene on human umbilical-vein endothelial cells (HUVECs), and to investigate whether there is an associated increased expression of some key regulators of the cell cycle. DESIGN: Cell culture for different incubation times. SETTING: University research laboratory. PATIENT(S): Sources of HUVECs. INTERVENTION(S): Measurement of cell proliferation, of protein levels of cyclin A, cyclin B1, cyclin D1, cyclin-dependent protein kinase (CDK) 2, CDK4, and p27(Kip1), and of messenger RNA expression of cyclin A, cyclin B1, and p27(Kip1). MAIN OUTCOME MEASURE(S): Cell proliferation was measured by the 3-(4.5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the bromo-2'-deoxyuridine assay, and the sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate assay. Changes in protein expression were measured by immunoblotting, and modifications in gene expression were measured by quantitative real-time polymerase chain reaction. RESULT(S): Both 1 nM and 10 nM of either E(2) or raloxifene achieved a similar increase in cell proliferation. The pure antiestrogen ICI 182780 only blocked the E(2)-induced proliferative effect. Western blot experiments detected an increase in the expression of only cyclin A and B1, and a decreasing trend for p27(Kip1). Enhancements in gene expression were observed in response to E(2) and raloxifene for cyclin A and B1. No significant changes were found for p27(Kip1). The ICI 182780 effectively abrogated the increased gene expression associated with E(2) for cyclin B1, but not for cyclin A. In contrast, ICI 182780 was ineffective in the case of raloxifene. CONCLUSION(S): Raloxifene increased the proliferation of HUVECs in association with enhanced gene expression of cyclins A and B1.

The risk for cardiovascular disease in women: from estrogens to selective estrogen receptor modulators.

Cardiovascular disease, a generic denomination including coronary heart disease (CHD), stroke, and venous thromboembolic disease (VTED), has shown sensitivity to estrogens. The relative protection of women as compared with men has nourished a debate about a possible protective role for estrogens, but the prejudicial effects detected in clinical trials has created confusion on the risk/benefit ratio induced by hormone administration. The hypothesis that agonists distinct to estrogens might improve the effects associated with estrogens is at the base of the increasing interest on the role of selective estrogen receptor modulators (SERMs). There is a lack of definitive clearcut clinical data on the effects of SERMs in CVD, although the available information suggests a neutral balance on CHD and stroke and an increase in risk similar to estrogens for VTED. Research on pathogenetic mechanisms concentrates in atherosclerosis as the main determinant of the arterial forms of the disease and in hypercoagulability as the counterpart for venous disease. The different experimental models used up to the present moment suggest that, compared with estrogens, SERMs play a less active protection against atherogenesis but do not increase vulnerability of unstable plaques. There is not a clear notion on the mechanisms promoted by SERMs to increase risk for VTED.

Estradiol counteracts oxidized LDL-induced asymmetric dimethylarginine production by cultured human endothelial cells.

OBJECTIVE: Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthase, is a novel cardiovascular risk factor produced by endothelial cells. ADMA levels are mainly regulated by the activity of dimethylarginine dimethylaminohydrolases (DDAH). Endothelial release of ADMA is increased in the presence of oxidized LDL cholesterol (oxLDL), whereas estrogens stimulate NO production by endothelial cells by increasing both expression and activity of NO synthase and by reducing ADMA levels. Thus, the aim of the present study was to evaluate the estradiol effects on the DDAH/ADMA/NO pathway in cultured human umbilical vein endothelial cells (HUVEC) exposed to LDL. METHODS: After 24 h of exposure to various treatments, culture medium was collected to measure NO production by using an amperometric sensor specific for NO, and to measure dimethylarginines by high-performance liquid chromatography (HPLC). DDAH-I and II mRNA expression and protein content were quantified by real-time PCR assay and immunoblotting, respectively. RESULTS: Exposure of HUVEC to 100 microg/mL oxLDL, but not to 100 microg/mL of native LDL (nLDL), reduced DDAH-II expression at both the mRNA as well as the protein levels, which in turn increased ADMA levels and reduced NO production. Estradiol (1 nM) alone increased DDAH-II mRNA and protein expression, which reduced ADMA levels and increased NO production. In cells exposed to estradiol in combination with either nLDL or oxLDL, levels of DDAH-II, ADMA, and NO were the same as those for estradiol alone. CONCLUSION: Estradiol completely reverses the effects induced by oxLDL on the DDAH/ADMA/NO pathway.

Therapeutic dosages of raloxifene do not modify myeloperoxidase and F2alpha-isoprostane levels in postmenopausal women.

We investigated the effect of a therapeutic dose of raloxifene on the plasma levels of myeloperoxidase and F2alpha-isoprostanes, two markers of oxidative stress recently described as reliable indicators of coronary heart disease. Contrary to changes described in the literature for estrogens (E), raloxifene did not modify the levels of either myeloproxidase or F2alpha-isoprostanes after 3 or 6 months of treatment.

Effects of phytoestrogens genistein and daidzein on prostacyclin production by human endothelial cells.

The molecular mechanisms of the vascular effects of phytoestrogens are poorly studied. Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase (COX) in endothelium. This study examine the effects of two phytoestrogens, the isoflavones genistein and daidzein, on prostacyclin production by cultured human umbilical vein endothelial cells (HUVECs) and the possible role of not only estrogen receptors but also both COX isoforms. The two phytoestrogens significantly increased prostacyclin release in a time- and dose-dependent (0.01-1 microM) manner, being higher than control after 24 h. Selective inhibitors of COX-1, SC-560 [5-(4-chlorophenyl)-1-(4-methoxypjenyl)-3-(trifluoromethyl)-1H-pyrazole], and COX-2, NS-398 (N-[2-(cyclohexyloxy)-4 nitrophenyl]-methanesulfonamide), were used to investigate the relative contribution of each enzyme. Both inhibitors decreased basal production of prostacyclin, but only COX-2 inhibition completely abolished the isoflavone-stimulated prostacyclin production. Phytoestrogens also increased COX-2 mRNA expression and protein content without affecting COX-1 levels. All these effects were mediated through estrogen receptor activation since treatment of cells with the estrogen receptor antagonist ICI 182780 [7alpha-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]-estra-1,3,5(10)-triene-3,17beta diol] completely abolished the isoflavone-induced increase in prostacyclin production, COX-2 mRNA expression, and COX-2 protein content. The results clearly support the hypothesis that genistein and daidzein increased HUVEC prostacyclin production through estrogen receptor-dependent mechanism, which involved the enhancement of COX-2 protein and activity.

Raloxifene promotes prostacyclin release in human endothelial cells through a mechanism that involves cyclooxygenase-1 and -2.

OBJECTIVE: To examine the effects of raloxifene on prostacyclin production by human umbilical vein endothelial cells (HUVEC) and to shed light on the molecular details of that action. DESIGN: Cell culture for 4, 8, 16, 24, and 48 hours. SETTING: University research laboratory. PATIENT(S): Source of HUVEC. INTERVENTION(S): Measurement of prostacyclin production and of protein levels and mRNA expression of cyclooxygenase (COX)-1 and -2. MAIN OUTCOME MEASURE(S): Prostacyclin production was measured by enzyme immunoassay, the mRNA expression of COX-1 was measured by quantitative real time-polymerase chain reaction, and the protein levels of COX-1 and -2 were measured by immunoblotting. RESULT(S): Raloxifene significantly increased prostacyclin release in a time- and dose-dependent manner, being higher than control after 24 hours. Raloxifene, at 0.1-10 nM, increased the mRNA expression of COX-1 and the protein content of both COX-1 as well as COX-2. All of these effects were independent of the classical pathway for estrogen receptor (ER) activation because the treatment of cells with the ER antagonist ICI 182780 did not eliminate any of the effects. Although treatment with either the selective COX-1 inhibitor SC-560 or the selective COX-2 inhibitor NS-398 significantly diminished prostacyclin release (20% +/- 5% and 24% +/- 7%, respectively), co-treatment with raloxifene and either SC-560 or NS-398 was followed by a smaller increase than that achieved by raloxifene alone. The nonselective COX inhibitor indomethacin, however, reduced prostacyclin production to 37% +/- 11% of control values. CONCLUSION(S): Raloxifene increased HUVEC prostacyclin release through a mechanism possibly distinct from the classical ER pathway and involving enhanced COX-1 and COX-2 expression and activity.

Raloxifene increases the capacity of serum to promote prostacyclin release in human endothelial cells: implication of COX-1 and COX-2.

OBJECTIVE: Prostacyclin is a potent vasodilator synthesized by two isoforms of cyclooxygenase in endothelium. The aim of this study was to investigate the effect of serum from postmenopausal women treated with raloxifene on prostacyclin production by human umbilical vein endothelial cells and on cyclooxygenases-1 and -2. DESIGN: Serum was collected from 21 women receiving 60 mg/day of raloxifene, at baseline and at 3 and 6 months. Human umbilical vein endothelial cells were exposed to serum for 24 hours, and prostacyclin production was evaluated in supernatants. Selective inhibitors of cyclooxygenases-1 and -2 (SC-560 and NS-398) were used to investigate the relative contribution of each enzyme. Protein expression for each enzyme was determined using Western blot assays. RESULTS: Prostacyclin production was increased by 30% (P < or = 0.001) when serum from women treated for 3 and 6 months was added. SC-560 reversed prostacyclin production but did not change baseline values. NS-398, in turn, reduced prostacyclin production using sera from baseline, 3 and 6 months. Cyclooxygenases-1 and -2 protein expression remained unaltered at each treatment step. CONCLUSIONS: Serum from women treated with raloxifene stimulated prostacyclin release from human umbilical vein endothelial cells in culture, an effect that seems mediated by increased cyclooxygenase-1 activity.

Validación del Nuevo Ensayo Tiempo de Bothrops atrox diluido (dBat) para la Detección de Anticoagulante Lúpico / Diluted Bothrops atrox time (dBat). A new assay to detect Lupus anticoagulant

Validar un nuevo ensayo para detección de anticoagulante lúpico, el cual emplea el veneno crudo definido Bohrops atrox (dBat), comparado con cuatro ensayos de coagulación fosfolípidodependientes: dRVVT, TTI y TNP. Material y Métodos: Se evaluó 70 muestras de plasma citratado de pacientes con diagnóstico clínico de LES, síndrome antifosfolípidico primario (SAFP) trombosis embolia y sospecha de trombosis, hospitalizados en el Hospital Nacional Guillermo Almenara Irigoyen durante Julio de 1998 y Marzo de 1999 . El dRVVT fue el análisis de referencia. Resultados : Hubo 30 muestras positivas por dBat y dRVVT y uno más de los restantes ensayos 27 casos fueron negativos y hubo 13 casos en los que los resultados obtenidos por el dBat fueron doscordantes respecto a los obtenidos por el dRVVT (p>0,05) De los casos con LES el 47.8 por ciento fueron positivos para AL, de los de SAFP el 71,4 por ciento de los casos de trombosis el 75 por ciento y de los casos con sospecha de trombosis el 47,8 por ciento.Conclusiones: Por la alta concordancia entre el dBat con el dBVVT y el correlato con la clínica, así como por poseer una mayor validez con respecto a los ensayos TTPa, TTI y TNP, el nuevo ensayo dBat puede ser empleado en la detección de AL.

Cooling Raw Milk: Change in the Spoilage Potential of Contaminating Pseudomonas.

A group of 80 Pseudomonas spp. strains isolated from raw milk shortly after milking was compared to another group of 81 obtained from the same sample after incubating it at 7°C for 3 days. Comparison of both collections of strains included growth rates at 7°C and 21°C and production of extracellular proteinase, lipase, and siderophores. The strains selected after cold incubation showed an average to-fold higher growth rate at 7°C, 1,000-fold more proteolytic activity, and 280-fold more lipolytic activity than those found before the incubation. At 21°C, however, they grew half as quickly as the strains isolated before the incubation. In all but one of the 161 Pseudomonas strains tested, there was some production of siderophores, and yields were only moderately increased in the strains obtained after incubation of the milk at 7°C. These changes in spoilage-related properties took place while global Pseudomonas counts increased only 13-fold. Distribution frequencies of the variables tested, their correlation coefficients, and regression models are shown.