ICI - CICS | Documentos por Auto-Depósito
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- IL-15 induces antigen-independent expansion and differentiation of human naive CD8+ T cells in vitroPublication . Alves, N L; Hooibrink, Berend; Arosa, FA; Van Lier, ReneRecent studies in mice have shown that although interleukin 15 (IL-15) plays an important role in regulating homeostasis of memory CD8+ T cells, it has no apparent function in controlling homeostatic proliferation of naive T cells. We here assessed the influence of IL-15 on antigen-independent expansion and differentiation of human CD8+ T cells. Both naive and primed human T cells divided in response to IL-15. In this process, naive CD8+ T cells successively down-regulated CD45RA and CD28 but maintained CD27 expression. Concomitant with these phenotypic changes, naive cells acquired the ability to produce interferon γ (IFN-γ) and tumor necrosis factor α (TNF-α), expressed perforin and granzyme B, and acquired cytotoxic properties. Primed CD8+ T cells, from both noncytotoxic (CD45RA-CD27+) and cytotoxic (CD45RA+CD27-) subsets, responded to IL-15 and yielded ample numbers of cytokine-secreting and cytotoxic effector cells. In summary, all human CD8+ T-cell subsets had the ability to respond to IL-15, which suggests a generic influence of this cytokine on CD8+ T-cell homeostasis in man. (Blood. 2003;102:2541-2546).
- 17 Beta-Estradiol and progesterone inhibit L-type Ca2+ current of rat aorta smooth muscle cellsPublication . Verde, Ignacio; Cairrão, Elisa; Carvas, João; Silva, António José Santos; Alvarez, EzequielSex hormones like 17ß-estradiol (ßES) and progesterone have shown rapid non-genomic vasodilator effects, which could be involved in the protection of cardiovascular system. However, the precise mechanism by which this effect occurs has not been elucidated yet, even if Ca2+ influx inhibition seems to be implicated. The aim of this study was to study the influence of ßES and progesterone on the L-type Ca2+ current measured by whole cell voltage-clamp in A7r5 cells. Voltage-operated Ca2+ currents were elicited by square-step voltage pulses and pharmacologically characterized as L-type currents by (-)-Bay K8644 (BAY) and nifedipine. Both ßES and progesterone (1-100 µM), rapidly and reversibly inhibited, in a concentration dependent manner, either non-stimulated or BAY-stimulated Ca2+ currents registered in A7r5 cells. These results suggest that ßES and progesterone inhibit L-type voltage-operated Ca2+ channels through a non-genomic pathway. Consequently, these hormones inhibit the Ca2+ entry into smooth muscle cells from rat aorta, an effect that can contribute for the protection of the cardiovascular system.
- Functional and phenotypic characterization of CD8+CD28+ and CD28- T cells in atopic individuals sensitized to Dermatophagoides pteronyssinusPublication . Lourenço, Olga; Fonseca, A. M.; Paiva, Artur; Arosa, F. A.; Barata, Luís TabordaCD8+ T suppressor cells may play a role in immunoregulation. Recent studies have characterized this population by the lack of the CD28 molecule. These CD8+CD28 T cells differ phenotypically and functionally from CD8 + CD28 + T cells. Little is known about CD8 + CD28 cells in atopy. Our aim was to analyze the phenotype and functional properties of CD8 + CD28T cells in atopic and non-atopic individuals.
- Determination of parathion in biological fluids by means of direct Solid Phase Microextraction.Publication . Gallardo, Eugenia; Barroso, Mário; Margalho, Cláudia; Cruz, Angelines; Vieira, Duarte Nuno; López-Rivadulla, ManuelA new and simple procedure for the determination of parathion in human whole blood and urine using direct immersion (DI) solid-phase microextraction (SPME) and gas chromatography/mass spectrometry (GC/MS) is presented. This technique was developed using only 100 ìL of sample, and ethion was used as internal standard (IS). A 65-ìm Carbowax/divinylbenzene (CW/DVB) SPME fibre was selected for sampling, and the main parameters affecting the SPME process such as extraction temperature, adsorption and desorption time, salt addition, agitation and pH effect were optimized to enhance the sensitivity of the method. This optimization was also performed to allow the qualitative determination of parathion’s main metabolite, paraoxon, in blood. The limits of detection and quantitation for parathion were 3 and 10 ng/mL for urine and 25 and 50 ng/mL for blood, respectively. For paraoxon, the limit of detection was 50 ng/mL in blood. The method showed linearity between the LOQ and 50 ìg/mL for both matrices, with correlation coefficients ranging from 0.9954 to 0.9999. Precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The mean absolute recoveries were 35.1% for urine and 6.7% for blood. Other parameters such as dilution of sample and stability were also validated. Its simplicity and the fact that only 100 ìL of sample is required to accomplish the analysis make this method useful in forensic toxicology laboratories to determine this compound in intoxications, and it can be considered an alternative to other methods normally used for the determination of this compound in biological media.
- Demographic, laboratory and clinical characterisation of adult portuguese asthmatic patientsPublication . Lourenço, Olga; Fonseca, A. M.; Barata, Luis TabordaAsthma is a heterogeneous chronic inflammatory condition characterised by reversible airway obstruction and hyperresponsiveness associated with underlying bronchial inflammation and structural changes. It represents an increasing health problem and is a huge burden on the patients, their families and society. The aim of the study was to characterise the adult asthmatic population attending a Hospital Allergy Clinic between the years of 2003 and 2006.
- Effects of trans- and cis-resveratrol on Ca2+ handling in A7r5 vascular myocytesPublication . Verde, Ignacio; Campos-Toimil, Manuel; Elies, Jacobo; Alvarez, Ezequiel; Orallo, FranciscoAlthough the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.
- Solid-phase extraction for gas chromatographic mass spectrometric determination of the veterinary drug xylazine in human blood.Publication . Gallardo, Eugenia; Barroso, Mário; Margalho, Cláudia; Devesa, N.; Pimentel, J.; Vieira, Duarte NunoThis paper presents a method for the determination of xylazine in whole blood using solid-phase extraction and gas chromatography–mass spectrometry. This technique required only 0.5 mL of sample, and protriptyline was used as internal standard (IS). Limits of detection and quantitation (LOQ) were 2 and 10 ng/mL, respectively. The method was found to be linear between the LOQ and 3.50 ìg/mL, with correlation coefficients higher than 0.9922. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The analyte was stable in the matrix for at least 18 h at room temperature and for at least three freeze/thaw cycles. Mean recovery, calculated at three concentration levels, was 87%. To the best of our knowledge, this is the first time that solid-phase extraction is used as sample preparation technique for the determination of this compound in biological media. Because of its simplicity and speed when compared to other extraction techniques, the herein described method can be successfully applied in the diagnosis of intoxications by xylazine.
- Potassium channels are involved in testosterone-induced vasorelaxation of human umbilical arteryPublication . Verde, Ignacio; Cairrão, Elisa; Álvarez, Ezequiel; Silva, António José SantosRecent studies have shown that testosterone induces relaxation of different arteries, although the mechanism of this action is still under debate. We investigated the involvement of potassium channels in this mechanism. Using standard organ bath techniques, rings of human umbilical arteries (HUA) without endothelium were contracted by serotonin (5-HT, 1 μM), histamine (10 μM) and potassium chloride (KCl, 30 and 60 mM), and the vasorelaxant effect of testosterone was analysed. Testosterone (100 μM) relaxed human umbilical arteries contracted with 5-HT (30.1±3.2%), histamine (55.1±2.6%), KCl 30 mM (52.9±8.3%) and KCl 60 mM (54.8±6.3%). Flutamide (10 μM), an inhibitor of classical intracellular testosterone receptor, and glibenclamide, an ATP-sensitive potassium-channels (KATP) inhibitor, did not influence the testosterone relaxant effect. 4-aminopyridine, a voltagesensitive potassium-channels (Kv) inhibitor, decreased the effect of testosterone on histamine- and 5-HT-contracted arteries. Tetraethylammonium (TEA), which inhibits Kv channels and large-conductance Ca2+-activated potassium channels (BKCa), decreased the effect of testosterone on KCl (60 mM)-contracted and 5-HT-contracted HUA. In conclusion, testosterone induces relaxation of HUA, and this effect does not appear to be mediated via a classic intracellular testosterone receptor-dependent mechanism. Our results suggest that this relaxation is partially mediated by activation of BKCa and KV channels. The involvement of these two channels in testosterone-relaxant mechanism is dependent on the pathways activated by the contractile agent used.
- STEAP1 is over-expressed in breast cancer and down-regulated by 17β-estradiol in MCF-7 cells and in the rat mammary glandPublication . Maia, C J; Socorro, Sílvia; Schmitt, Fernando; Santos, CeciliaSix transmembrane epithelial antigen of the prostate 1 (STEAP1) was identified as a prostate-specific cell-surface antigen over-expressed in prostate cancer, and in human cancer cell lines obtained from several other tissues. Its cell surface location in all tumor types analyzed so far, and its absence in most vital organs in humans, turned STEAP1 into a potential target for anti-tumor immunotherapy. This study provides experimental evidence that STEAP1 is also over-expressed in human breast cancer cases, and in normal breast tissue adjacent to breast tumors, where it is localized in the cell membrane of epithelial cells. It is also demonstrated that STEAP1 transcription correlates negatively with estrogen receptor (ER) immunoreactivity, and positively with tumor grading in breast cancer cases. As estrogens are involved in breast cancer onset and progression, the response of STEAP1 to 17beta-estradiol (E2) was investigated in the mammary gland of rats, and in the human breast cancer cell line, MCF-7. These experiments demonstrated that STEAP1 is down-regulated by E2 in both models. The mechanisms underlying the STEAP1 response to E2 in vitro were further investigated in MCF-7 cells, and the results obtained suggest an effect mediated by the membrane-bound ERalpha (mbERalpha).
- Development and validation of an analytical method for the simultaneous determination of cocaine and its main metabolite, benzoylecgonine, in human hair by gas chromatography/mass spectrometry.Publication . Gallardo, Eugenia; Dias, M.; Vieira, Duarte Nuno; Queiroz, João; López-Rivadulla, ManuelA new, simple and rapid procedure has been developed and validated for the determination of cocaine and its main metabolite, benzoylecgonine, in human hair samples. After extraction from within the hair matrix by a mixture of methanol/hydrochloric acid (2:1) at 65-C for 3 h, and sample cleanup by mixed-mode solid-phase extraction (SPE), the extracts were analyzed by gas chromatography/ mass spectrometry (GC/MS), after derivatization with N-methyl-N-(trimethylsilyl)trifluoroacetamide with 5% chlorotrimethylsilane. Using a sample size of only 20mg of hair, limits of detection (LODs) and quantitation (LOQs) were, respectively, 20 and 50 pg/mg for cocaine, and 15 and 50 pg/mg for benzoylecgonine, achieving the cut-off values proposed by the Society of Hair Testing for the analysis of these compounds in hair. The method was found to be linear (weighing factor of 1/x) between the LOQ and 20 ng/mg for both compounds, with correlation coefficients ranging from 0.9974 to 0.9996 for cocaine; and from 0.9981 to 0.9994 for benzoylecgonine. Intra- and interday precision and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The sample cleanup step presented a mean absolute recovery greater than 90% for both compounds. The developed method may be useful in forensic toxicology laboratories for the analysis of cocaine and benzoylecgonine in hair samples, taking into account its speed (only 3 h are required for the extraction of the analytes from within the matrix, whereas 5 h or even overnight extractions have been reported) and the low limits achieved (using a single quadrupole mass spectrometer, which is available in most laboratories).