Patrocinio Molinero

Interaction of vasoactive intestinal peptide (VIP) with rat lymphoid cells

Receptors for vasoactive intestinal peptide (VIP) have been characterized in rat lymphoid cells. The interaction of [125I] VIP with blood mononuclear cells was rapid, reversible, specific and saturable. At apparent equilibrium, the binding of [125I] VIP was competitively inhibited by native VIP in the 0.01-100 nM range concentration. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.050 +/- 0.009 nM and a low binding capacity (2.60 +/- 0.28 fmol/10(6) cells), and a low-affinity class with a Kd = 142 +/- 80 nM and a high binding capacity (1966 +/- 330 fmol/10(6) cells). Secretin, glucagon, insulin and somatostatin did not show any effect at a concentration as high as 100 nM. With spleen lymphoid cells, stoichiometric studies were performed. The binding data were compatible with the existence of two classes of receptors: a high-affinity class with a Kd = 0.100 +/- 0.033 nM and a low binding capacity (4.60 +/- 1.07 fmol/10(6) cells), and low-affinity class with a Kd = 255 +/- 110 nM and high binding capacity (2915 +/- 1160 fmol/10(6) cells). With thymocytes, no binding was obtained under different conditions.

Interaction of vasoactive intestinal peptide (VIP) with human peripheral blood lymphocytes: specific binding and cyclic AMP production.

VIP binding sites and cyclic AMP production by the peptide have been studied in human blood mononuclear cells before and after selective depletion of or enrichment for T-lymphocytes, B-lymphocytes-K-NK cells and monocytes. The specifically bound 125I-labelled VIP correlated significantly with the presence of B-lymphocytes and/or cells of K-NK system. The stoichiometric data were compatible with the existence of two classes of binding sites. T-lymphocytes and monocytes did not show binding of the tracer. The cyclic AMP production stimulated by VIP correlated significantly with the presence of B-lymphocytes and/or K-NK cells.

Melatonin biosynthesis in the thymus of humans and rats

Abstract.Melatonin is an indoleamine widely distributed in the evolution that shows a great functional versatility, playing an important role as a transmitter of photoperiodic information and exhibiting antioxidant, oncostatic, anti-aging and immunomodulatory properties. In vertebrates, this molecule is produced by the pineal gland and other extrapineal sites. The present study was carried out to investigate the presence of melatonin in thymus and the possibility of an endogenous melatonin synthesis in this organ, in which T cells are matured. In this work, we demonstrate in humans and rats that thymus contains melatonin, expresses the mRNAs encoding N-acetyltransferase and hydroxyindol-O-methyltransferase, the two key enzymes of the melatonin synthesis, and has this biosynthetic machinery activated. In addition, rat thymocytes cultured for 24 h exhibited high levels of melatonin. The results presented here suggest that human and rat thymuses are able to synthesize melatonin, which could have intracrine, autocrine and paracrine functions.

Role of early cell-free DNA levels decrease as a predictive marker of fatal outcome after severe traumatic brain injury.

INTRODUCTION Circulating cell-free DNA levels are increased after trauma injury. This increase is higher since the first hours after trauma and may be related with primary outcome. A sensitive and reliable biomarker for patients at higher risk is needed to identify these patients to initiate early intervention. In this way, circulating DNA may be a possible biological marker after severe TBI. MATERIALS AND METHODS We investigated DNA plasma concentrations after severe traumatic brain injury and during the next 96 h in the Intensive Care Unit (ICU) by real time PCR. 65 patients suffering severe TBI were included in the study. RESULTS Cell-free DNA levels were considerably higher in patients samples compared with voluntary control ones. After the following four days we observed a 51% decrease during the first 24h and a 71% fall from 48 h. TBI population was stratified for the primary outcome (survivors/non-survivor) and DNA levels decrease ratio was calculated for the first 48 h. A higher decrease in the survivors from 0 h to 24h compared with the non-survivors was found. A cut-off point of 1.95 ratio was established for the detection of the highest proportions of patients after the TBI that will not survive after the injury with a sensitivity of 70% and specificity of 66%. CONCLUSIONS In summary we showed that severe TBI is associated with elevated cf-DNA levels and we propose that cf-DNA decrease during the first 24h may predict patient outcome.

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes : significance in IL-2 production

Abstract.Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE2 in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE2-diminished IL-2 production. The decrease in IL-2 production after PGE2 and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE2 in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT1 antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology.

Evidence for melatonin synthesis in the rat brain during development

Abstract:  Melatonin production is not restricted to the pineal gland. Several extrapineal sources of this indole such as retina, Harderian gland, and immune system are well documented. Melatonin of pineal origin is not present in the rat at early stages of development. To assess the potential capacity of local melatonin synthesis by the immature brain and to gain insight into the relationship between melatonin production by the brain (without the pineal gland) and pineal gland during rat development, the melatonin content as well as the expression and activity of the melatonin‐synthesizing enzymes, N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT), were studied at fetal and postnatal stages. Moreover, melatonin‐membrane receptor (MT1) expression was also analyzed. Both, the expression and activity of NAT and HIOMT were found in the brain with significant day/night differences in enzymes activities. Additionally, melatonin content was detected in all stages showing day/night differences depending on the stage of development. The brain nocturnal melatonin content was higher than diurnal content on postnatal day 16 and in adult rats which is in accordance with the pineal melatonin synthesis. To investigate the origin of this brain melatonin, pinealectomized rats were used and we found that the developing brain produced its own melatonin. Also, MT1 expression was detected in brain during development. These results demonstrate that, when the pineal is not yet producing melatonin, there is melatonin synthesis by the brain that could be used as protection from free radical damage and/or could exert some actions through MT1 receptors.

Melatonin is responsible for the nocturnal increase observed in serum and thymus of thymosin α1 and thymulin concentrations: observations in rats and humans

This paper shows that melatonin regulates both thymosin alpha1 and thymulin production as well as the expression of the prothymosin alpha gene. The results revealed the following facts: (a) The concentrations of thymosin alpha1 in both serum and thymus of rat showed a nyctohemeral profile with peak values late at night and basal values during the day. The concentrations of thymulin in rat serum also showed a 24-h rhythm with an increase in their values at night. This rhythmical character for thymosin alpha1, and thymulin was also found in the human serum. (b) Rats injected with melatonin during the day exhibited a significant increase in the concentrations of both peptides. Moreover, continuous light exposure on the animals at daytime and pinealectomy cause a decrease in thymosin a1 and thymulin concentrations with regards to those found in control rats. (c) Melatonin regulates the expression of the prothymosin alpha gene, analyzed by Northern blot. These results suggest that melatonin may be involved in the regulation of immune functions by increasing the thymic peptides production.

Circadian variations in the rat serum total antioxidant status: Correlation with melatonin levels

ABSTRACT: In this paper, we show for the first time, a nyctohemeral rhythm in serum total antioxidant status (TAS) in rats which parallels the 24‐H melatonin cycle. Both TAS and melatonin in rat serum exhibited 24 hr variations with nocturnal peak values at 05.00 hr and low basal values during the day. When rats were maintained under light exposure (>500 lux) from 20.00 h to 05.00 hr, serum TAS was significantly reduced when compared with control rat killed in darkness. Moreover, when animals were maintained under continuous light exposure for 5 days and killed at 05.00 hr, serum TAS exhibited an additional decrease when compared with control rats. Since administering exogenous melatonin also increased TAS in the rat serum, results suggest that melatonin may be relevant in terms of participating in the antioxidative capacity of the rat serum.

Involvement of Nuclear Receptors in the Enhanced IL-2 Production by Melatonin in Jurkat Cells

Abstract: This report shows that melatonin enhances IL‐2 production by Jurkat cells via a nuclear receptor‐mediated mechanism. Jurkat cells express nuclear (RZRα, RORα1, and RORα2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL‐2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates the IL‐2 production. These results show that the expression of the nuclear melatonin receptor is sufficient for melatonin to activate IL‐2 production by Jurkat cells.

Melatonin synthesis and melatonin‐membrane receptor (MT1) expression during rat thymus development: role of the pineal gland

Abstract:  To gain insight into the relationship between thymus and pineal gland during rat development, the melatonin content as well as the activity and expression of the two key enzymes for melatonin biosynthesis, i.e. N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT), were studied in the thymus at fetal and postnatal stages. Moreover, melatonin‐membrane receptor (MT1) expression was also analyzed. We found both the expression and activity of thymic NAT and HIOMT at 18 days of fetal life. Additionally, there is production of melatonin in the thymus as well as MT1 expression at this fetal age. These results show values higher in day‐time than at night‐time. The pineal gland begins to produce significant levels of melatonin around postnatal day 16, and this synthesis shows a circadian rhythm with high values during the dark period; therefore the nocturnal serum melatonin may inhibit thymic melatonin production. To document this, we report an increased melatonin content of the thymus in pinealectomized rats compared with sham‐pinealectomized. In conclusion, these results show, for the first time, the presence of the biosynthetic machinery of melatonin and melatonin production in developing rat thymus and that the pineal gland may regulate this process.