RESUMEN
Saliva measurements serve as a noninvasive tool for clinically monitoring newborns (NB) and children, a vulnerable population with promising potential for both research and clinical practice. Saliva acts as a repository for various inflammatory biomarkers involved in diverse biological functions. Particularly for children, it offers numerous advantages when compared to plasma and urine sampling. Nevertheless, there is a significant knowledge gap regarding detectable levels of cytokines in the saliva of newborns and children, as well as studies aiming to assess the relationship of this content with physiological and pathological processes. OBJECTIVES: To characterize the levels of 11 inflammatory mediators (IFNg, IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17, TNF, and VEGF) in saliva samples from NB on the first and second day of hospitalization in the Neonatal Intensive Care Unit (NICU). METHOD: Exploratory study, descriptive, nested within a primary clinical, observational, and prospective study, conducted in the NICU of a public hospital in São Paulo, Brazil. Demographic data and vital signs were recorded in the clinical records of 90 NB, and five saliva samples from 5 NB were collected between the first and second day of life (D1-D2) at approximately 8-hr intervals (8-9 am, 4-5 pm, and 11-12 pm). Saliva samples were used for the measurement of 11 cytokines (IFNg, IL1b, IL2, IL4, IL6, IL8, IL10, IL12, IL17, TNF, and VEGF). RESULTS: Five NBs participated in this exploratory study, and the vital signs showed variability from the first (D1) to the second day (D2) of hospitalization, variability similar to that of the total population of the primary study. The presence and levels of the 11 cytokines were detected in the saliva samples, as well as a statistical correlation between 10 cytokines (IFNg, IL1b, IL2, IL4, IL6, IL10, IL12, IL17, TNF, and VEGF) and vital signs. CONCLUSIONS: The novelty of measuring inflammatory mediators in saliva samples from hospitalized NBs in the NICU is highlighted, providing support and new perspectives for the development of clinical and experimental research and an opportunity for developing and implementing new salivary biomarkers in different population segments.
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Biomarcadores , Citocinas , Unidades de Cuidado Intensivo Neonatal , Saliva , Humanos , Saliva/química , Recién Nacido , Biomarcadores/análisis , Biomarcadores/metabolismo , Masculino , Femenino , Citocinas/análisis , Citocinas/metabolismo , Estudios Prospectivos , Brasil , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/análisis , HospitalizaciónRESUMEN
Nuclear factor-kappa B (NFKB), a pivotal player in inflammatory responses, is constitutively expressed in the pineal gland. Corticosterone inhibits pineal NFKB leading to an enhancement of melatonin production, while tumor necrosis factor (TNF) leads to inhibition of Aa-nat transcription and the production of N-acetylserotonin in cultured glands. The reduction in nocturnal melatonin surge favors the mounting of the inflammatory response. Despite these data, there is no clear evidence of the ability of the pineal gland to recognize molecules that signal infection. This study investigated whether the rat pineal gland expresses receptors for lipopolysaccharide (LPS), the endotoxin from the membranes of Gram-negative bacteria, and to establish the mechanism of action of LPS. Here, we show that pineal glands possess both CD14 and toll-like receptor 4 (TLR4), membrane proteins that bind LPS and trigger the NFKB pathway. LPS induced the nuclear translocation of p50/p50 and p50/RELA dimers and the synthesis of TNF. The maximal expression of TNF in cultured glands coincides with an increase in the expression of TNF receptor 1 (TNFR1) in isolated pinealocytes. In addition, LPS inhibited the synthesis of N-acetylserotonin and melatonin. Therefore, the pineal gland transduces Gram-negative endotoxin stimulation by producing TNF and inhibiting melatonin synthesis. Here, we provide evidence to reinforce the idea of an immune-pineal axis, showing that the pineal gland is a constitutive player in the innate immune response.
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Receptores de Lipopolisacáridos/metabolismo , FN-kappa B/metabolismo , Glándula Pineal/metabolismo , Receptor Toll-Like 4/metabolismo , Análisis de Varianza , Animales , Extractos Celulares/química , Células Cultivadas , Ensayo de Cambio de Movilidad Electroforética , Femenino , Inmunidad Innata/fisiología , Inmunohistoquímica , Receptores de Lipopolisacáridos/biosíntesis , Receptores de Lipopolisacáridos/genética , Lipopolisacáridos/metabolismo , Masculino , Glándula Pineal/citología , Glándula Pineal/inmunología , ARN Mensajero , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Técnicas de Cultivo de Tejidos , Receptor Toll-Like 4/biosíntesis , Receptor Toll-Like 4/genéticaRESUMEN
Exposure to fungicide ziram (zinc dimethyldithiocarbamate) has been associated with increased incidence of Parkinson's disease (PD). We recently demonstrated that the intranasal (i.n.) administration of sodium dimethyldithiocarbamate (NaDMDC, a more soluble salt than ziram) induces PD-like behavioral and neurochemical alterations in mice. We now investigated the putative neuroprotective effects of melatonin on behavioral dificits and neurochemical alterations induced by i.n. NaDMDC. Melatonin treatment (3, 10 or 30 mg/kg, i.p.) was given 1 h before NaDMDC administration (1 mg/nostril) during 4 consecutive days and we evaluated early (up to 7 days) and late (up to 35 days) NaDMDC-induced behavioral and neurochemical alterations. Melatonin treatment protected against early motor and general neurological impairments observed in the open field and neurological score of severity, respectively, and late deficits in rotarod test. Melatonin prevented the NaDMDC-induced alterations in the striatal tyrosine hydroxylase immunocontent. Melatonin also protected against increased levels of oxidative stress markers (4-hydroxynonenal and 3-nitrotyrosine) in the striatum, as well as the NaDMDC-induced increase of 4-hydroxynonenal and TNF, markers of oxidative stress and inflammation, respectively, in the olfactory bulb. These results further detail the mechanisms underlying NaDMDC toxicity and demonstrate the neuroprotective effects of melatonin against the neuronal damage induced by NaDMDC.
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Encéfalo/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Melatonina/farmacología , Fármacos Neuroprotectores/farmacología , Síndromes de Neurotoxicidad/prevención & control , Administración Intranasal , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/fisiopatología , Dimetilditiocarbamato , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Masculino , Ratones , Actividad Motora/efectos de los fármacos , Síndromes de Neurotoxicidad/etiología , Síndromes de Neurotoxicidad/metabolismo , Síndromes de Neurotoxicidad/fisiopatología , Estrés Nitrosativo/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacosRESUMEN
Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappaB) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappaB translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mum) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappaB pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.
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Células Endoteliales/metabolismo , Lipopolisacáridos/inmunología , Melatonina/farmacología , Óxido Nítrico/metabolismo , Análisis de Varianza , Animales , Aorta/efectos de los fármacos , Aorta/metabolismo , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Masculino , FN-kappa B/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Ratas , Ratas Wistar , Vasodilatación/efectos de los fármacosRESUMEN
BACKGROUND: The alterations in neurological and neuroendocrine functions observed in the autism spectrum disorder (ASD) involves environmentally dependent dysregulation of neurodevelopment, in interaction with multiple coding gene defects. Disturbed sleep-wake patterns, as well as abnormal melatonin and glucocorticoid secretion, show the relevance of an underlying impairment of the circadian timing system to the behavioral phenotype of ASD. Thus, understanding the mechanisms involved in the circadian dysregulation in ASD could help to identify early biomarkers to improve the diagnosis and therapeutics as well as providing a significant impact on the lifelong prognosis. OBJECTIVE: In this review, we discuss the organization of the circadian timing system and explore the connection between neuroanatomic, molecular, and neuroendocrine responses of ASD and its clinical manifestations. Here we propose interconnections between circadian dysregulation, inflammatory baseline and behavioral changes in ASD. Taking into account, the high relevancy of melatonin in orchestrating both circadian timing and the maintenance of physiological immune quiescence, we raise the hypothesis that melatonin or analogs should be considered as a pharmacological approach to suppress inflammation and circadian misalignment in ASD patients. STRATEGY: This review provides a comprehensive update on the state-of-art of studies related to inflammatory states and ASD with a special focus on the relationship with melatonin and clock genes. The hypothesis raised above was analyzed according to the published data. CONCLUSION: Current evidence supports the existence of associations between ASD to circadian dysregulation, behavior problems, increased inflammatory levels of cytokines, sleep disorders, as well as reduced circadian neuroendocrine responses. Indeed, major effects may be related to a low melatonin rhythm. We propose that maintaining the proper rhythm of the circadian timing system may be helpful to improve the health and to cope with several behavioral changes observed in ASD subjects.
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Trastorno del Espectro Autista/fisiopatología , Trastornos Cronobiológicos/complicaciones , Ritmo Circadiano , Melatonina/fisiología , Trastorno del Espectro Autista/complicaciones , Humanos , Trastornos del Sueño-Vigilia/complicacionesRESUMEN
Phosphoinositide signaling pathway orchestrates primordial molecular and cellular functions in both healthy and pathologic conditions. Phosphatidylinositol-5-phosphate 4-kinase type 2 lipid kinase (PIP4K2) family, which compromises PIP4K2A, PIP4K2B and PIP4K2C, has drawn the attention in human cancers. Particularly in hematological malignancies, PIP4K2A was already described as an essential protein for a malignant phenotype, although the clinical and biological impact of PIP4K2B and PIP4K2C proteins have not being explored in the same extent. In the present study, we investigated the impact on clinical outcomes and gene network of PIP4K2A, PIP4K2B and PIP4K2C mRNA transcripts in acute myeloid leukemia (AML) patients included in The Cancer Genome Atlas (2013) study. Our results indicate that PIP4K2A and PIP4K2C, but not PIP4K2B, mRNA levels were significantly reduced in AML patients assigned to the favorable risk group (p < 0.05) and low levels of PIP4K2A and PIP4K2C positively affect clinical outcomes of AML patients (p < 0.05). Gene set enrichment analyses indicate that the expression of PIP4K2 genes is associated with biological process such as signal transduction, metabolism of RNA and genomic instability related-gene sets. In summary, our study provides additional evidence of the involvement of members of the PIP4K2 family, in particular PIP4K2A and PIP4K2C, in AML.
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Predisposición Genética a la Enfermedad , Leucemia Mieloide Aguda/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , ARN Mensajero/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mutación , Análisis de Supervivencia , Adulto JovenRESUMEN
Energy deficiency and dysfunction of the Na,K-ATPase are common consequences of many pathological insults. Glutamate through cyclic GMP and cyclic GMP-dependent protein kinase (PKG) has been shown to stimulate alpha(2/3)-Na,K-ATPase activity in the central nervous system. Thus, a slight impairment of this pathway may amplify the disruption of ion homeostasis in the presence of a non-lethal insult. We investigate the effect of aging (4, 12 and 24 months) on the glutamate-cyclic GMP-PKG modulation of alpha1, alpha(2/3)-Na,K-ATPase activity in rat cerebellum and the stimulation of the glutamate-cyclic GMP-PKG pathway at different levels. Cyclic GMP levels and alpha(2/3)-Na,K-ATPase activity were progressively decreased from 4 and 24 month-old animals. However, PKG basal activity was reduced between 4 and 12 months, and no additional change was observed at 24 months. The ability of 8-Br-cyclic GMP to stimulate PKG activity was only reduced between 12 and 24 months. Moreover, glutamate or 8-Br-cyclic GMP promoted a smaller increase of alpha(2/3)-Na,K-ATPase activity at 24 months, when compared to 4 and 12 months. In spite of the age-related reduced basal levels of cyclic GMP, the production induced by CO or NO was not age-related. Finally, inhibition of PKG activation by KT5823 revealed a lower sensitivity of the enzyme at the older age. Taken together, these data show that basal age-related decline in sodium pump activity is a consequence of changes in different steps of the cyclic GMP-PKG pathway. On the other hand, age-related reduction in glutamate positive modulation of cerebellar alpha(2/3)-Na,K-ATPase is linked to a defective PKG signaling pathway.
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Envejecimiento/metabolismo , Cerebelo/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Adaptación Fisiológica/fisiología , Animales , Activación Enzimática , Técnicas In Vitro , Masculino , RatasRESUMEN
Shiftwork-induced sleep deprivation and circadian disruption probably leads to an increase in the production of cytokines and dysregulation of innate immune system, respectively. This project aims evaluating changes in salivary IL-1 beta, cortisol, and melatonin in night workers. Method. Two day and three night healthy workers participated in this study. Sleep was evaluated by actimetry and activity protocols. Saliva was collected at waking and bedtime the last workday and the following two days-off and was analyzed by ELISA. Results. Neither sleep duration nor efficiency showed any association with salivary IL-1beta. IL-1beta levels were higher at waking than at bedtime during working days for all workers, but only one day and one night-worker maintained this pattern and hormone rhythms during days off. For this night worker, melatonin levels were shifted to daytime. A second one presented clear alterations in IL-1beta and hormone rhythms on days-off. Conclusions. Our preliminary results suggest that night work can disturb the variation pattern of salivary IL-1beta. No association of this variation with sleep was observed. It seems that disruption in hormone rhythms interfere with salivary IL-1beta production. IL- 1beta production pattern seems to be maintained when rhythms are present, in spite of a shift in melatonin secretion.
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Hidrocortisona/metabolismo , Interleucina-1beta/metabolismo , Melatonina/metabolismo , Sueño/fisiología , Tolerancia al Trabajo Programado/fisiología , Ritmo Circadiano , Humanos , Saliva/metabolismoRESUMEN
We have previously shown that melatonin influences the development of alpha8 nicotinic acetylcholine receptor (nAChR) by measurement of the acetylcholine-induced increase in the extracellular acidification rate (ECAR) in chick retinal cell cultures. Cellular differentiation that takes place between DIV (days in vitro) 4 and DIV 5 yields cells expressing alpha8 nAChR and results in a significant increase in the ECAR acetylcholine-induced. Blocking melatonin receptors with luzindole for 48h suppresses the development of functional alpha8 nAChR. Here we investigated the time window for the effect of melatonin on retinal cell development in culture, and whether this effect was dependent on an increase in the expression of alpha8 nAChR. First, we confirmed that luzindole was inhibiting the effects of endogenous melatonin, since it increases 2-[(125)I] iodomelatonin (23pM) binding sites density in a time-dependent manner. Then we observed that acute (15, 60min, or 12h) luzindole treatment did not impair acetylcholine-induced increase in the ECAR mediated by activation of alpha8 nAChR at DIV 5, while chronic treatment (from DIV 3 or DIV 4 till DIV 5, or DIV 3.5 till DIV 4.5) led to a time-dependent reduction of the increase in the acetylcholine-induced ECAR. The binding parameters for [(125)I]-alpha-bungarotoxin (10nM) sites in membrane were unaffected by melatonin suppression that started at DIV 3. Thus, melatonin surges in the time window that occurs at the final stages of chick retinal cell differentiation in culture is essential for development of the cells expressing alpha8 nAChR subtype in full functional form.
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Melatonina/metabolismo , Isoformas de Proteínas/metabolismo , Receptores Nicotínicos/metabolismo , Retina/metabolismo , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Embrión de Pollo , Regulación del Desarrollo de la Expresión Génica , Radioisótopos de Yodo/metabolismo , Isoformas de Proteínas/genética , Receptores de Melatonina/antagonistas & inhibidores , Receptores de Melatonina/metabolismo , Receptores Nicotínicos/genética , Retina/citología , Retina/efectos de los fármacos , Factores de Tiempo , TriptaminasRESUMEN
BACKGROUND: Endothelial cells are of great interest for cell therapy and tissue engineering. Understanding the heterogeneity among cell lines originating from different sources and culture protocols may allow more standardized material to be obtained. In a recent paper, we showed that adrenalectomy interferes with the expression of membrane adhesion molecules on endothelial cells maintained in culture for 16 to 18 days. In addition, the pineal hormone, melatonin, reduces the adhesion of neutrophils to post-capillary veins in rats. Here, we evaluated whether the reactivity of cultured endothelial cells maintained for more than two weeks in culture is inversely correlated to plasma melatonin concentration. METHODOLOGY/PRINCIPAL FINDINGS: The nocturnal levels of melatonin were manipulated by treating rats with LPS. Nocturnal plasma melatonin, significantly reduced two hours after LPS treatment, returned to control levels after six hours. Endothelial cells obtained from animals that had lower nocturnal melatonin levels significantly express enhanced adhesion molecules and iNOS, and have more leukocytes adhered than cells from animals that had normal nocturnal levels of melatonin (naïve or injected with vehicle). Endothelial cells from animals sacrificed two hours after a simultaneous injection of LPS and melatonin present similar phenotype and function than those obtained from control animals. Analyzing together all the data, taking into account the plasma melatonin concentration versus the expression of adhesion molecules or iNOS we detected a significant inverse correlation. CONCLUSIONS/SIGNIFICANCE: Our data strongly suggest that the plasma melatonin level primes endothelial cells "in vivo," indicating that the state of the donor animal is translated to cells in culture and therefore, should be considered for establishing cell banks in ideal conditions.
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Ritmo Circadiano/fisiología , Células Endoteliales/citología , Melatonina/sangre , Animales , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Molécula 1 de Adhesión Intercelular/metabolismo , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/metabolismo , Lipopolisacáridos/farmacología , Neutrófilos/citología , Óxido Nítrico Sintasa de Tipo II/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Ratas , Ratas Wistar , Factores de TiempoRESUMEN
The temporal organization of mammals presents a daily adjustment to the environmental light/dark cycle. The environmental light detected by the retina adjusts the central clock in the suprachiasmatic nuclei, which innervate the pineal gland through a polysynaptic pathway. During the night, this gland produces and releases the nocturnal hormone melatonin, which circulates throughout the whole body and adjusts several bodily functions according to the existence and duration of darkness. We have previously shown that during the time frame of an inflammatory response, pro-inflammatory cytokines, such as tumor necrosis factor-alpha, inhibit while anti-inflammatory mediators, such as glucocorticoids, enhance the synthesis of melatonin, interfering in the daily adjustment of the light/dark cycle. Therefore, injury disconnects the organism from environmental cycling, while recovery restores the light/dark information to the whole organism. Here, we extend these observations by evaluating the effect of a mild restraint stress, which did not induce macroscopic gastric lesions. After 2 h of restraint, there was an increase in circulating corticosterone, indicating activation of the hypothalamus-pituitary-adrenal (HPA) axis. In parallel, an increase in melatonin production was observed. Taking into account the data obtained with models of inflammation and stress, we reinforce the hypothesis that the activity of the pineal gland is modulated by the state of the immune system and the HPA axis, implicating the darkness hormone melatonin as a modulator of defense responses.
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Sistema Inmunológico/fisiopatología , Glándula Pineal/inmunología , Glándula Pineal/fisiopatología , Estrés Psicológico/fisiopatología , Animales , Ritmo Circadiano/fisiología , Corticosterona/sangre , Ambiente , Humanos , Mediadores de Inflamación/metabolismo , Melatonina/metabolismo , Restricción Física , Estrés Psicológico/sangreRESUMEN
We evaluated whether changes in protein content and activity of PP-1 and PP-2A were the mechanism underneath the basal age-related reduction in alpha(2/3)-Na,K-ATPase activity in rats cerebella and whether this occurred through the cyclic GMP-PKG pathway. PP1 activity, but not its expression, increased with age, whereas PP-2 was not changed. The activity of alpha(2/3)-Na,K-ATPase varied with age, and there was a negative association between the PP-1 and alpha(2/3)-Na,K-ATPase activities. In young rats, the inhibition of PP-1 and PP-2A by okadaic acid (OA) increased in a dose-dependent manner alpha(1)- and alpha(2/3)-Na,K-ATPase, but had no effect on Mg-ATPase activity. A direct stimulation of PKG with 8-Br-cyclic GMP did not surmount the effect of OA. This analogue of cyclic GMP inhibited PP-1 activity only, indicating that at least part of the increase in alpha(1)- and alpha(2/3)-Na,K-ATPase activity induced by OA was mediated by the cyclic GMP-PKG-PP-1 cascade. Taking into account that PP1 inhibition increased alpha(2/3)-Na,K-ATPase activity, we propose that an age-related increase in PP-1 activity due to a decrease in cyclic GMP-PKG modulation plays a role for the age-related reduction of alpha(2/3)-Na,K-ATPase activity in rat cerebellum.
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Envejecimiento/metabolismo , Cerebelo/enzimología , Proteína Fosfatasa 1/metabolismo , Transducción de Señal/fisiología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Activación Enzimática , Masculino , Ratas , Ratas WistarRESUMEN
Melatonin, known as the hallmark of darkness, is released during the dark by pineal glands and retina of adult animals. In neonates melatonin is secreted in a tonic manner by extra-pineal sources, including retina. Here we show that melatonin modulates the density and functional response of nicotinic acetylcholine receptors (nAChRs) in central and peripheral nervous system. Melatonin effect is restricted to α-bungarotoxin sensitive nAChRs in all models tested: rat sympathetic nerve terminals, cerebellum, and chick retina. In addition, it is shown that in adult animals, where the pineal gland releases melatonin in a rhythm manner, this hormone imposes a daily rhythm in the cholinergic function, whereas during the development of the retina, when melatonin is produced in a tonic manner, it is essential for the appearance of function nAChRs sensitive to α-bungarotoxin. This common pattern of action on different cell models that express α-bungarotoxin-sensitive receptors probably reflects a more general mechanism of regulation of these receptors.