RESUMEN
Dendritic cell (DC) cancer vaccines have shown limited clinical benefit. Thus, the identification of signals and molecular pathways that potentiate the immunogenicity of DCs has become a major challenge in cancer research. Our studies demonstrate that triiodothyronine endows DCs with enhanced ability to stimulate cytotoxic T-cell responses with implications in DC-based immunotherapy.
RESUMEN
Sulphated esters of the flavonoids sulphated quercetin 3,7,3',4'-tetrasulphated (QTS) and quercetin 3-acetyl-7,3,4'-trisulphate (ATS), isolated from Flaveria bidentis, have demonstrated anticoagulant and antiplatelet properties. In this study, we examined if both compounds affected the expression of the procoagulant tissue factor (TF) induced by lipopolysaccharide (LPS) on human monocyte. Monocytes were pretreated with different concentrations of each flavonoid (0.1-500 µM), followed by a 4h incubation with LPS in order to induce TF expression. Results of the TF expression showed different behaviors for the two flavonoids studied. A slight inhibitory effect on the TF expression was detected at a QTS concentration of 0.1 µM, but from 1 µM onwards a significant inhibitory effect that remained up to 500 µM could be observed. In contrast, ATS induced a poor inhibitory effect on TF expression at all concentrations tested. These results suggest that QTS has another antithrombotic property, to be added to its already renowned ability as an anticoagulant and antiplatelet compound.
Asunto(s)
Fibrinolíticos/farmacología , Flaveria/química , Monocitos/efectos de los fármacos , Extractos Vegetales/farmacología , Quercetina/análogos & derivados , Tromboplastina/metabolismo , Fibrinolíticos/aislamiento & purificación , Humanos , Lipopolisacáridos , Monocitos/metabolismo , Quercetina/aislamiento & purificación , Quercetina/farmacologíaRESUMEN
OBJECTIVE: Turner syndrome (TS) is an indication for GH therapy in spite of the modest growth response. Somatic growth depends not only on GH insulin-like growth factor I (IGF-I) axis but also on thyroid hormone (TH) status. We have previously reported that supraphysiological IGF-I levels diminished TH actions in rat tissues by reducing the nuclear TH receptor (TR). GH treatment to TS patients induces high IGF-I levels and therefore a reduction of TH action in tissues may be expected. We aimed at evaluating the effect of GH therapy in TS girls on peripheral TH action. DESIGN AND PATIENTS: We set up a reverse transcription-polymerase chain reaction (RT-PCR) for TR mRNA estimation in peripheral blood mononuclear cells (PBMC) and compared TR mRNA levels from 10 normal, 10 TS and 10 TS girls under GH therapy (0.33 mg/kg/week for 0.5-2 years). MEASUREMENTS: After RNA extraction from PBMC, TR and beta-actin mRNAs were coamplified by RT-PCR. In addition serum biochemical markers of TH action were measured: thyrotropin (TSH), sex hormone binding globulin (SHBG), osteocalcin (OC), beta-crosslaps (beta-CL), iodothyronines by electrochemiluminescency and IGF-I by immunoradiometric assay (IRMA) with extraction. RESULTS: TR mRNAs from PBMC were reduced in TS patients under GH treatment. In turn, serum TSH, OC, beta-CL and IGF-I were increased while SHBG was reduced by GH treatment in TS patients. CONCLUSIONS: GH treatment reduced TR expression in PBMC and biochemical serum markers of TH action. These results suggest that GH treatment in TS patients impair peripheral TH action at tissue level and prompt a role in the reduced growth response to the therapy.
Asunto(s)
Hormona del Crecimiento/uso terapéutico , Hormonas Tiroideas/sangre , Síndrome de Turner/tratamiento farmacológico , Actinas/genética , Análisis de Varianza , Biomarcadores/sangre , Estudios de Casos y Controles , Niño , Colágeno/sangre , Depresión Química , Femenino , Estudios de Seguimiento , Humanos , Factor I del Crecimiento Similar a la Insulina/análisis , Osteocalcina/sangre , Fragmentos de Péptidos/sangre , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Globulina de Unión a Hormona Sexual/análisis , Receptores alfa de Hormona Tiroidea/genética , Receptores beta de Hormona Tiroidea/genética , Tirotropina/sangre , Triyodotironina/sangre , Síndrome de Turner/sangreRESUMEN
Triiodothyronine (T3) exerts most of its effect through nuclear thyroid hormone receptors (TR) which bind mainly as heterodimers with retinoid-X receptors (RXR) to thyroid hormone response elements (TRE) in target genes. It is well known that the synergistic interaction of T3 and glucocorticoids has a role on the synthesis of growth hormone in rat pituitary cell lines and in the T3-induced metamorphosis in amphibians. Glucocorticoids increased mRNAs of T3-regulated hepatic genes. Our laboratory reported increased specific metabolic actions of T3 in rat liver by Dexamethasone (Dex) through a mechanism involving an up-regulation of the maximal binding capacity of TR. In this study we further explored the participation of TR in the molecular mechanism of the Dex-induced increase on liver T3-specific metabolic action. Dex administration to adrenalectomized rats induced an increase of liver TRbeta1 protein and mRNA. Nuclear run-on assay revealed that Dex up-regulated the TR gene transcriptional rate. Transfection assay in COS-7 cells indicated that Dex increased the transcriptional activity of the TRbeta1 promoter. Electrophoretic mobility shift assay demonstrated that Dex induced the binding of additional proteins related to or neighboring the DNA sequence of a glucocorticoid receptor (GR) binding (GRE) half-site in the TRbeta1 promoter. Evidences for an interaction of GR on the TRbeta1 promoter have been obtained. Moreover, the specificity of the GR binding to GRE was determined not only by the GRE DNA sequence, but also by the interaction of the GR with other transacting factors bound to sequences flanking the GRE.
Asunto(s)
Antiinflamatorios/farmacología , Dexametasona/farmacología , Hígado/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Receptores beta de Hormona Tiroidea/genética , Transcripción Genética/efectos de los fármacos , Adrenalectomía , Animales , Secuencia de Bases , Células COS , Chlorocebus aethiops , Hígado/metabolismo , Masculino , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Receptores beta de Hormona Tiroidea/metabolismoRESUMEN
The involvement of atrial natriuretic peptide (ANP) in the regulation of thyroid gland is supported by the presence of high-affinity ANP receptors and the identification of the peptide in thyroid follicular cells. The aim of this work was to study the action of ANP on parameters of thyroid hormone biosynthesis and analyze the intracellular mechanism of the ANP action in cultured bovine thyroid follicles. The addition of ANP (0.1-10 nM) to the culture medium for 24 h inhibited the TSH (thyroid-stimulating hormone)-stimulated iodide uptake with a maximal inhibition at 1 nM ANP. When thyrocytes were incubated with 10 nM ANP the inhibitory effect slightly increased from 24 to 72 h. Thyroglobulin (Tg) mRNA expression was reduced by 1 and 10 nM ANP. After 24 h of treatment with the cGMP analogue, N(2),2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate [(Bu)(2)cGMP] (0.1 and 1 mM), an inhibition of iodide uptake and Tg mRNA expression was obtained, evidencing a cGMP-mediated inhibitory signal in the thyroid cell. A reduction of the cAMP production was induced by incubation of thyroid follicles with 1 and 10 nM ANP for 24 h. Under a similar treatment the cGMP accumulation was increased only by 10 nM ANP. The inhibitory effect of ANP on Tg mRNA level was reverted in the presence of pertussis toxin, an inhibitor of the G(i)-protein-mediated reduction of the adenylate cyclase activity. These results indicate an inhibitory action of ANP on parameters of thyroid hormone biosynthesis. A G(i)-protein-mediated reduction of the cAMP production seems to be the main factor involved in the ANP action although a role of the cGMP pathway should not be discarded specially at high ANP levels.
Asunto(s)
Factor Natriurético Atrial/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Yoduros/metabolismo , Tiroglobulina/genética , Glándula Tiroides/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Bovinos , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Toxina del Pertussis/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/efectos de los fármacos , Glándula Tiroides/citología , Glándula Tiroides/metabolismo , Hormonas Tiroideas/biosíntesisRESUMEN
In this investigation, we studied the effects of Momany peptide (GHRP-5), on somatotroph secretory activity. Acute and chronic administration of GHRP-5 provokes a significant release of growth hormone that can be closely correlated with ultrastructural changes in somatotroph populations. After 3,5 and 7 days of GHRP-5 treatment, two somatotroph cell subpopulations coexist. One of them has an enhanced secretory activity and the other presents a quiescent appearance. Therefore, pituitary growth hormone content was not affected in the first seven days of GHRP-5 treatment. After 14 days, there was a significant depletion of growth hormone pituitary content coincident with the highest levels of serum growth hormone. These results concur with the surge of a new hyperactive somatotroph subtype characterised by numerous immature secretory granules that are discharged bypassing the maturation step. Acute and chronic treatments caused no changes in somatotroph cell density, the area immunostained for growth hormone and the levels of total mRNA for transcription factor pit-1. The results of pituitary cell cultures incubated with specific blockers for different signalling pathways demonstrated an involvement of the phospholipase C-inositol phosphate system in GHRP-5 stimulated somatotroph secretion. GHRP-5 treatment enhanced significantly the release of growth hormone, thereby eliciting ultrastructural modifications in somatotrophs that can be correlated with an increased secretory activity devoid of cell density changes.
Asunto(s)
Hormona del Crecimiento/metabolismo , Oligopéptidos/farmacología , Adenohipófisis/efectos de los fármacos , Animales , Recuento de Células , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Inmunohistoquímica , Masculino , Microscopía Inmunoelectrónica , Adenohipófisis/citología , Adenohipófisis/metabolismo , Pirrolidinonas/farmacología , ARN Mensajero/metabolismo , Radioinmunoensayo , Ratas , Ratas Wistar , Vesículas Secretoras/efectos de los fármacos , Vesículas Secretoras/ultraestructura , Transducción de Señal , Tinción con Nitrato de Plata , Hormona Liberadora de Tirotropina/farmacología , Factor de Transcripción Pit-1 , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
The effects of the tri-iodothyronine (T(3)) secreted by thyroid cells on the growth of the thyrocyte are poorly known. In this study we analyzed the effects of T(3) on the proliferation of bovine thyroid follicles in primary culture previously depleted of endogenous T(3). Cellular deoxiribonucleic acid (DNA) synthesis, determined by [(3)H]thymidine incorporation, was stimulated by T(3) (0.1-5.0 nM) for 24 h in a concentration-dependent fashion with a maximal effect at 1.0 nM T(3) (P<0.01). This T(3) action was time-dependent when assayed from 12 to 72 h. The induction of mitogenic activity was corroborated by the increase in proliferating cell nuclear antigen (PCNA) measured by Western blot analysis. PCNA increased after treatment with T(3) (0.1-5.0 nM) in a concentration-dependent manner. Since T(3) modifies the activity of growth factors whose actions are mainly mediated by tyrosine kinase (TK) activation in diverse cellular types, we assayed the effects of genistein, a general TK inhibitor, and tyrphostin A25, a specific epidermal growth factor (EGF)-receptor (EGFR)-dependent TK activity inhibitor, on the proliferative effects of T(3). The T(3)-induced [(3)H]thymidine incorporation was inhibited by both agents in a concentration-dependent manner. A significant increase in the total TK activity measured in cellular protein extracts was induced by 0.5 and 1.0 nM T(3) (P<0.001). Tyrosine phosphorylation of the EGFR was also stimulated by T(3) (P<0.001) with no change in the EGFR expression as determined by Western blot analysis. Both, the T(3)-stimulated [(3)H]thymidine incorporation and the TK activity were inhibited by a anti-mouse EGF antibody. These results lead us to propose that T(3) could operate as a proliferative agent in bovine thyroid cells through a mechanism involving an autocrine/paracrine EGF/EGFR-dependent regulation.
Asunto(s)
Receptores ErbB/metabolismo , Glándula Tiroides/metabolismo , Triyodotironina/farmacología , Análisis de Varianza , Animales , Biomarcadores/análisis , Western Blotting , Bovinos , División Celular/efectos de los fármacos , Células Cultivadas , ADN/biosíntesis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Receptores ErbB/antagonistas & inhibidores , Genisteína/farmacología , Fosforilación , Antígeno Nuclear de Célula en Proliferación/análisis , Proteínas Tirosina Quinasas/metabolismo , Estimulación Química , Timidina Quinasa/antagonistas & inhibidores , Glándula Tiroides/efectos de los fármacos , Tirfostinos/farmacologíaRESUMEN
Insulin-like growth factor I (IGF-I) downregulates growth hormone (GH) expression in pituitary cell cultures. However, in vivo different results were found depending on the experimental protocol used. We determined the kinetics of changes of pituitary and serum GH concentrations after subcutaneous IGF-I administration (240 microg/100 g body weight) to rats every 12 h for various periods. These parameters were correlated with changes in the somatotroph cell population. A significant increase in serum GH was registered at 6 h after IGF-I injection. At this time, some somatotroph cells exhibited ultrastructurally signs of high secretory activity, whereas adjacent somatotroph cells showed a quiescent appearance with sizeable stores of secretory granules. In contrast, serum GH levels remained unchanged at 1, 2 and 12 h after each IGF-I injection. Pituitary GH concentrations were comparable to control levels during the first 48 h and declined significantly at 72 h and 96 h of IGF-I treatment. After these prolonged periods of time of treatment, the size and extension of organelles involved in protein synthesis decreased and mature secretory granules in the cytoplasm increased significantly in GH-secreting cells. The somatotroph cell density remained unchanged even at 96 h of treatment. In conclusion, our results suggest that periodical IGF-I administration to rats does not inhibit GH secretion. Interestingly, IGF-I injections induced early and significant increases in serum GH levels. This result may be a consequence of a temporary stimulatory action on somatotroph cells concurrent with increased secretory activity.
Asunto(s)
Sangre/efectos de los fármacos , Hormona del Crecimiento/metabolismo , Factor I del Crecimiento Similar a la Insulina/farmacología , Hipófisis/efectos de los fármacos , Animales , Citoplasma/metabolismo , Regulación hacia Abajo , Inmunohistoquímica , Cinética , Masculino , Microscopía Electrónica , Hipófisis/ultraestructura , Radioinmunoensayo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
Tri-iodothyronine (T3) is known to be involved in the regulation of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis. In previous studies we demonstrated that IGF-I and GH reduced the metabolic response to T3 measured as the activity of two T3-dependent enzymes, mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) in cultured rat liver cells. In this study we analysed in vivo the effect of IGF-I administered to rats on the activity of alpha-GPD and ME. IGF-I (240 micrograms/100 g body weight (BW) every 12 h for 48 h) significantly diminished alpha-GPD (P < 0.01) and ME (P < 0.05) activities. Serum basal glucose concentration was not significantly modified 12 h after the administration of recombinant human IGF-I (240 and 480 micrograms/100 g BW every 12 h for 48 h). Under similar conditions, no significant change in serum total thyroxine (TT4) concentration was observed, although free thyroxine (FT4) was diminished (P < 0.02) and total T3 (TT3) was increased (P < 0.03). To explore the participation of the nuclear thyroid hormone receptor (THR) in the mechanism of IGF-I action we measured the maximal binding capacity and the affinity constant (Ka) of THR by Scatchard analysis, and concentrations of messenger RNAs (mRNAs) that code for the isoforms of THR present in the liver (beta 1, alpha 1 and alpha 2) by Northern blot. IGF-I (240 micrograms/100 g BW every 12 h for 48 h) significantly reduced maximal binding capacity to 37% of the control value (P < 0.01) without changes in the Ka. beta 1, alpha 1 and alpha 2 THR mRNAs were significantly reduced (P < 0.01) by 120-480 micrograms/100 g BW IGF-I administration every 12 h for 48 h. Time-course studies indicated that this effect was obtained 12 h after the administration of 240 micrograms/100 g BW IGF-I (P < 0.05). These results indicate that IGF-I administration to rats diminishes the metabolic thyroid hormone action in the liver by a mechanism that involves, at least in part, a reduction in the number of THRs and in their level of expression.
Asunto(s)
Factor I del Crecimiento Similar a la Insulina/farmacología , Hígado/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Hormonas Tiroideas/metabolismo , Animales , Células Cultivadas , Citosol/efectos de los fármacos , Citosol/enzimología , Retroalimentación , Glicerolfosfato Deshidrogenasa/metabolismo , Hígado/efectos de los fármacos , Malato Deshidrogenasa/metabolismo , Masculino , Mitocondrias Hepáticas/efectos de los fármacos , Mitocondrias Hepáticas/enzimología , Unión Proteica , Ratas , Ratas Wistar , Tiroxina/metabolismo , Triyodotironina/metabolismoRESUMEN
Nitric oxide (NO) has been proposed as an intracellular signal in the thyroid. The NO effect on function and morphology of bovine thyroid follicles in culture was analyzed by using the NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Both NO donors induced a concentration-dependent NO release measured by the nitrite accumulation in the culture medium. The SNP (10 to 500 micromol/L) treatment for 24 hours significantly inhibited the uptake, organification and transport of iodide in a concentration-dependent manner. When SNP (50 micromol/L) was withdrawn from the culture medium after 24 hours' incubation, iodide uptake and organification were partially recovered at 24 hours and reached the control value at 48 hours, indicating a reversible effect of SNP. A possible involvement of cyanide in the SNP inhibitory effect was excluded because incubation of follicles with potassium cyanide (KCN) at concentrations estimated to be present in the medium (40 and 80 micromol/L) for 24 hours did not modify iodide uptake and organification. The GSNO (10 to 500 micromol/L) treatment for 24 hours also reduced the iodide uptake, organification and transport in a concentration-dependent manner. A significant inhibition of iodide organification was induced after incubation with 1000 micromol/L of N2, 2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate ([Bu]2cGMP). Morphological evaluation by light microscopy revealed that the incubation with NPS or GSNO (500 micromol/L) produced cellular dispersion with loss of follicular cell aggregates that was evident at 96 hours exposure. Cell viability was not altered by 10-500 micromol/L SNP or GSNO (80% to 85%). We concluded that long-term NO exposure induces functional and morphological modifications compatible with a loss of differentiation in thyroid follicles. These observations further support a role of NO in the regulation of the thyroid function.
Asunto(s)
Yoduros/metabolismo , Donantes de Óxido Nítrico/farmacología , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Bovinos , Células Cultivadas , Medios de Cultivo , GMP Dibutiril Cíclico/farmacología , Glutatión/análogos & derivados , Glutatión/farmacología , Óxido Nítrico/biosíntesis , Nitritos/metabolismo , Nitroprusiato/farmacología , Compuestos Nitrosos/farmacología , Cianuro de Potasio/farmacología , S-Nitrosoglutatión , Glándula Tiroides/metabolismoRESUMEN
Triiodothyronine (T3) is involved in the regulation of the growth hormone-insulin-like growth factor I (GH-IGF-I) axis. In this study we investigated the effect of GH and IGF-I on the metabolic response of T3 in target tissues by evaluating the activity of two T3-dependent liver enzymes: mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) and cytosolic malic enzyme (ME) in rat hepatocytes in primary culture. Growth hormone (35 nmol/l) as well as IGF-I (0.5 mumol/l) reduced alpha-GPD and ME activities (p < 0.01) compared to the control group. Timecourse studies indicated that IGF-I (1.5 mumol/l) significantly decreased alpha-GPD and ME activities (P < 0.01) after 24 h, whereas the effect of GH (35 nmol/l) was recorded only after 36 h (p < 0.01). This delayed effect of GH compared to IGF-I suggested the possibility that the effect of GH could be mediated by IGF-I synthesis. To test this hypothesis, the effect of GH on the two enzyme activities was studied in the presence of anti-IGF-I antibodies. A gradual recovery of alpha-GPD and ME activities (p < 0.01) was observed in the presence of GH (35 nmol/l) plus increasing concentrations of anti-IGF-I antiserum. The maximal alpha-GPD and ME activities attained after the incubation of the liver cells with 1 mumol/l T3, a concentration high enough to fully saturate the nuclear T3 receptors for 24 h, were lowered significantly by 1.0 mumol/l IGF-I (p < 0.01). This finding suggests that the IGF-I effect might be independent of the saturation of the nuclear T3 receptors. In conclusion, in cultured rat hepatocytes, GH and IGF-I reduced the metabolic response of T3 evaluated by two liver T3-dependent enzyme activities. The effect of GH was mediated at least in part by IGF-I.