Identification of Inflammatory Mediators in Saliva Samples From Hospitalized Newborns: Potential Biomarkers?

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.

IL-10-induced STAT3/NF-κB crosstalk modulates pineal and extra-pineal melatonin synthesis.

Immune-pineal axis activation is part of the assembly of immune responses. Proinflammatory cytokines inhibit the pineal synthesis of melatonin while inducing it in macrophages by mechanisms dependent on nuclear factor-κB (NF-κB) activation. Cytokines activating the Janus kinase/signal transducer and activator of transcription (STAT) pathways, such as interferon-gamma (IFN-γ) and interleukin-10 (IL-10), modulate melatonin synthesis in the pineal, bone marrow (BM), and spleen. The stimulatory effect of IFN-γ upon the pineal gland depends on STAT1/NF-κB interaction, but the mechanisms controlling IL-10 effects on melatonin synthesis remain unclear. Here, we evaluated the role of STAT3 and NF-κB activation by IL-10 upon the melatonin synthesis of rats' pineal gland, BM, spleen, and peritoneal cells. The results show that IL-10-induced interaction of (p)STAT3 with specific NF-κB dimmers leads to different cell effects. IL-10 increases the pineal's acetylserotonin O-methyltransferase (ASMT), N-acetylserotonin, and melatonin content via nuclear translocation of NF-κB/STAT3. In BM, the nuclear translocation of STAT3/p65-NF-κB complexes increases ASMT expression and melatonin content. Increased pSTAT3/p65-NF-κB nuclear translocation in the spleen enhances phosphorylated serotonin N-acetyltransferase ((p)SNAT) expression and melatonin content. Conversely, in peritoneal cells, IL-10 leads to NF-κB p50/p50 inhibitory dimmer nuclear translocation, decreasing (p)SNAT expression and melatonin content. In conclusion, IL-10's effects on melatonin production depend on the NF-κB subunits interacting with (p)STAT3. Thus, variations of IL-10 levels and downstream pathways during immune responses might be critical regulatory factors adjusting pineal and extra-pineal synthesis of melatonin.

Rat resistance to rheumatoid arthritis induction as a function of the early-phase adrenal-pineal crosstalk.

Chronic inflammatory diseases are triggered by causal stimuli that might occur long before the appearance of the symptoms. Increasing evidence suggests that these stimuli are necessary but not always sufficient to induce the diseases. The murine model of type II collagen emulsified in Freund's incomplete adjuvant (collagen-induced arthritis) to induce rheumatoid arthritis (RA) follows this pattern as some animals do not develop the chronically inflamed phenotype. Considering that in the immune-pineal axis (IPA) theory adrenal-pineal cross-talk adjusts early phases of inflammatory processes, we investigated whether differences in IPA activation could explain why some animals are resistant (RES) while others develop RA. We observed a similar increase in 6-sulfatoxymelatonin (aMT6s) excretion from day 3 to 13 in both RES and RA animals, followed by a significant decrease in RA animals. This pattern of aMT6s excretion positively correlated with plasma corticosterone (CORT) in RES animals. Additionally, RA animals presented a lower aMT6s/CORT ratio than saline-injected or RES animals. Plasmatic levels of tumour necrosis factor were similar in both groups, but interleukin (IL)-1ß and monocyte chemotactic protein 1 (MCP-1) levels were lower in RES compared to RA animals. IL-2 and IL-4 were decreased in RES animals compared to saline-injected animals. The aMT6s/CORT ratio inversely correlated with the paw thickness and the inflammatory score (levels of IL-1ß, MCP-1, IL-2 and IL-4 combined). Thus, adrenocortical-pineal positive interaction is an early defence mechanism for avoiding inflammatory chronification. KEY POINTS: Immune-pineal axis imbalance is observed in early-phase rheumatoid arthritis development. Only resistant animals present a positive association between adrenal and pineal hormones. The 6-sulfatoxymelatonin/corticosterone ratio is decreased in animals that develop rheumatoid arthritis. The inflammatory score combining the levels of nocturnal interleukin (IL)-1ß, monocyte chemotactic protein 1, IL-2 and IL-4 presents a very strong positive correlation with the size of inflammatory lesion. The 6-sulfatoxymelatonin/corticosterone ratio presents a strong negative correlation with the inflammatory score and paw oedema size.

MEL-Index: Estimation of Tissue Melatonin Levels Using Gene Expression Data.

Melatonin synthesis by extrapineal sources adjusts physiological and pathophysiological processes in several types of cells and tissues. As measuring locally produced melatonin in fresh tissues might be a challenge due to limited material availability, we created a simple predictive model, the MEL-Index, which infers the content of tissue melatonin using gene expression data. The MEL-Index can be a powerful tool to study the role of melatonin in different contexts. Applying the MEL-Index method to RNA-seq datasets, we have shed light into the clinical relevance of melatonin as a modulator tumor progression and lung infection due to COVID-19. The MEL-Index combines the z-normalized expressions of ASMT (Acetylserotonin O-Methyltransferase), last enzyme of the biosynthetic pathway, and CYP1B1 (cytochrome P450 family enzyme), which encodes the enzyme that metabolizes melatonin in extrahepatic tissues. In this chapter, we describe the steps for calculating the MEL-Index.

LPS-induced immunomodulation and hormonal variation over time in toads.

The inflammatory response is a complex process that relies on interactions among multiple endocrine and immune modulators. Studies incorporating time-related and integrative endocrine and immune responses to an immune challenge might shed light on the characterization of the phases of the inflammatory response in anurans. The present study investigated time-related changes (1, 3, 6, and 18 h post-challenge) in plasma corticosterone (CORT), melatonin (MEL) and testosterone (T) levels, phagocytosis percentage (PP), plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) following a lipopolysaccharide (LPS) immune challenge in Rhinella diptycha toads. Our results showed the response to LPS injection was characterized by increased CORT, PP, BKA, and NLR, with a concomitant decrease in plasma MEL and T. Increased CORT was more pronounced at 6 and 18 h, while increased NLR was observed only 18 h post-LPS injection. Meanwhile, plasma MEL and T decreased independently of the time post-LPS injection. Additionally, toads in better body condition showed higher BKA and PP in the LPS-treated group, regardless of the time postinjection. Our results show that toads (R. diptycha) were sensitive to the LPS challenge, mounting an inflammatory response, which started quickly (after 1 h) and developed over time and was influenced by body condition. These results demonstrate a time-related hormonal and immune variation as a consistent pattern of activation of the immune system, as well as of hypothalamic-pituitary-adrenal/interrenal and immune-pineal axes following an immune challenge more deeply studied in mammals, suggesting the evolutionary conservation of the regulatory mechanisms for tetrapod vertebrates.

Neuroprotective effects of melatonin against neurotoxicity induced by intranasal sodium dimethyldithiocarbamate administration in mice.

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.

STAT1-NFκB crosstalk triggered by interferon gamma regulates noradrenaline-induced pineal hormonal production.

Melatonin production by pineal glands is modulated by several immune signals. The nuclear translocation of nuclear factor kappa-B (NFκB) homodimers, lacking transactivation domains, once induced by lipopolysaccharide (LPS) or tumor necrosis factor (TNF), inhibits the expression of Aanat gene and the synthesis of noradrenaline (NA)-induced melatonin. Interferon gamma (IFN-γ), on the other hand, increases melatonin synthesis. Furthermore, this cytokine activates the signal transducer as well as the activator of transcription 1 (STAT1) pathway, which was never evaluated as a melatonin synthesis modulator before. Reports demonstrated that IFN-γ might also activate NFκB. The present study evaluated the role of STAT1-NFκB crosstalk triggered by IFN-γ regarding the regulation of NA-induced pineal glands' hormonal production. Moreover, IFN-γ treatment increased NA-induced Aanat transcription, in addition to the synthesis of N-acetylserotonin (NAS) and melatonin. These effects were associated with STAT1 nuclear translocation, confirmed by the co-immunoprecipitation of STAT1 and Aanat promoter. Pharmacological STAT1 enhancement augmented NA-induced Aanat transcription as well as NAS and melatonin production. Additionally, IFN-γ induced the nuclear translocation of RelA-NFκB subunits. The blockade of this pathway prevented IFN-γ effects on the pineal function. The present data show that STAT1 and NFκB crosstalk controls melatonin production through a synergistic mechanism, disclosing a new integrative mechanism regarding pineal hormonal activity control.

PIP4K2A and PIP4K2C transcript levels are associated with cytogenetic risk and survival outcomes in acute myeloid leukemia.

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.

Night work effects on salivary cytokines TNF, IL-1ß and IL-6.

Shift work is unavoidable in modern societies, but at the same time disrupts biological rhythms and contributes to social distress and disturbance of sleep, health and well-being of shift workers. Shift work has been associated with some chronic diseases in which a chronic inflammatory condition may play a role. However, few studies investigating the association of cytokine and other inflammation markers with shift workers have been published in recent years. In this study we evaluated the effects of permanent night work on the production of tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), IL-6 and melatonin in saliva. Another aim was to demonstrate the benefit of the use of salivary cytokines for studies in chronobiology, since it is an easy and non-invasive method that allows for sampling at several times. Thirty-eight healthy male workers, being 21 day workers and 17 night workers, agreed to participate in this study. Sleep was evaluated by actigraphy and activity protocols. Saliva was collected during three workdays approximately at the middle of the work shift and at bed and wake times of the main sleep episode. Saliva samples were then analyzed by enzyme-linked immunosorbent assay to measure TNF, IL-1ß, IL-6 and melatonin levels, and the results were submitted to non-parametric statistical analysis. The use of saliva instead of blood allowed for a greater number of samples from the same subjects, allowing identifying alterations in the daily production patterns of salivary cytokines TNF, IL-1ß and IL-6 that probably are linked to night work. Salivary TNF and IL-1ß levels were similar for day and night workers, with higher daily production after awakening, in the morning hours for day workers and in the afternoon for night workers. Both groups presented a significant daily variation pattern of these two cytokines. Day and night workers produced similar amounts of salivary IL-6. Nevertheless, the daily variation pattern observed among day workers, with a peak after awakening, was absent among night workers. Thus, in our study, night workers showed partially adjusted daily variation patterns for salivary TNF and IL-1ß, not seen for salivary IL-6. Results for salivary IL-6 could be better explained as a consequence of circadian disruption due to permanent night work. Our results suggest that the whole circadian system, including clocks and pineal gland, is involved in regulating cytokine profile in shift workers and that a coordinated production of these cytokines, important for an adequate inflammatory response, could be disturbed by shift work. The distinct effects that shift work may have on different cytokines could give some cues about the mechanisms involved in this association.

Interplay among steroids, body condition and immunity in response to long-term captivity in toads.

Stressful experiences can promote harmful effects on physiology and fitness. However, stress-mediated hormonal and immune changes are complex and may be highly dependent on body condition. Here, we investigated captivity-associated stress effects, over 7, 30, 60, and 90 days on plasma corticosterone (CORT) and testosterone (T) levels, body index, and innate immunity (bacterial killing ability and phagocytosis of peritoneal cells) in toads (Rhinella icterica). Toads in captivity exhibited elevated CORT and decreased T and immunity, without changes in body index. The inter-relationships between these variables were additionally contrasted with those obtained previously for R. schneideri, a related species that exhibited extreme loss of body mass under the same captive conditions. While T and phagocytosis were positively associated in both species, the relationship between CORT and bacterial killing ability was dependent on body index alterations. While CORT and bacterial killing ability were positively associated for toads that maintained body index, CORT was negatively associated with body index in toads that lost body mass over time in captivity. In these same toads, body index was positively associated with bacterial killing ability. These results demonstrate that steroids-immunity inter-relationships arising from prolonged exposure to a stressor in toads are highly dependent on body condition.

Expression of the Circadian Clock Gene BMAL1 Positively Correlates With Antitumor Immunity and Patient Survival in Metastatic Melanoma.

INTRODUCTION: Melanoma is the most lethal type of skin cancer, with increasing incidence and mortality rates worldwide. Multiple studies have demonstrated a link between cancer development/progression and circadian disruption; however, the complex role of tumor-autonomous molecular clocks remains poorly understood. With that in mind, we investigated the pathophysiological relevance of clock genes expression in metastatic melanoma. METHODS: We analyzed gene expression, somatic mutation, and clinical data from 340 metastatic melanomas from The Cancer Genome Atlas, as well as gene expression data from 234 normal skin samples from genotype-tissue expression. Findings were confirmed in independent datasets. RESULTS: In melanomas, the expression of most clock genes was remarkably reduced and displayed a disrupted pattern of co-expression compared to the normal skins, indicating a dysfunctional circadian clock. Importantly, we demonstrate that the expression of the clock gene aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) positively correlates with patient overall survival and with the expression of T-cell activity and exhaustion markers in the tumor bulk. Accordingly, high BMAL1 expression in pretreatment samples was significantly associated with clinical benefit from immune checkpoint inhibitors. The robust intratumoral T-cell infiltration/activation observed in patients with high BMAL1 expression was associated with a decreased expression of key DNA-repair enzymes, and with an increased mutational/neoantigen load. CONCLUSION: Overall, our data corroborate previous reports regarding the impact of BMAL1 expression on the cellular DNA-repair capacity and indicate that alterations in the tumor-autonomous molecular clock could influence the cellular composition of the surrounding microenvironment. Moreover, we revealed the potential of BMAL1 as a clinically relevant prognostic factor and biomarker for T-cell-based immunotherapies.

Behavioral fever decreases metabolic response to lipopolysaccharide in yellow Cururu toads (Rhinella icterica).

Ectothermic vertebrates develop behavioral fever in response to bacterial products, with potential corresponding metabolic costs associated with immune stimulation. Although behavioral fever has been described in several taxa under laboratory conditions, some important questions regarding metabolic response to bacterial products at different temperatures and effectiveness of behavioral fever remain open. Many ectotherms, such as nocturnal anurans, may be active in the field at environmental conditions that restrict thermoregulation during the immune response. How does the metabolic response to bacterial products under ecologically relevant but unfavorable thermal field conditions compare to that measured in fever thermal preferendum? Additionally, are there differences in the partitioning of metabolic costs associated with immune stimulation and Arrhenius effect (biochemical reactions rate) at normal versus fever thermal preferendum? We compared the energy expenditure untreated and LPS-treated yellow Cururu toads (Rhinella icterica) at temperature corresponding to field activity during winter nights, and at normal and fever thermal preferendum. It was hypothesized that the metabolic response to LPS would be proportionally lower at higher body temperatures. To test these hypotheses, we measured temperature in the field during night using agar models, as well as normal and fever thermal preferendum of the toads within a thermal gradient. Subsequently, we measured the toad's metabolic rates at mean agar models temperature, as well as at normal and fever thermal preferendum. Lastly, we calculated the Metabolic response to LPS as the ratio between MRLPS/MRSaline in each of these mean temperatures. Our results show that metabolic rates do not increase in response to LPS at the agar models temperature typical of the winter nights under which theses toads maintain reproductive activity. Moreover, LPS treatment increased the metabolic costs relative to Arrhenius effects at normal thermal preferendum but not at fever thermal preferendum. In this way, metabolic response to LPS was comparative lower at fever than normal thermal preferendum in yellow Cururu toads.

Captivity effects on immune response and steroid plasma levels of a Brazilian toad (Rhinella schneideri).

Stressors can increase plasma glucocorticoid (GC) levels and decrease plasma androgen levels in different species of vertebrates. GCs can have immune-enhancing or immunosuppressive effects, which are dependent upon stress duration and intensity. The worldwide decline in amphibian populations is strongly linked to an array of different stressors. The impacts of stress on GCs, androgens, and the immune response are important to clarify and should lead to the better development of conservation strategies. The present study in adult male toads of Rhinella schneideri investigated the effects of captivity (7, 30, and 60 days) on plasma corticosterone (CORT) and plasma testosterone (T), as well as innate immune responses, specifically humoral and cell mediated responses, as indicated by bacterial killing ability (BKA) and phagocytosis by peritoneal cells, respectively. Captivity increased CORT threefold and decreased T versus controls. CORT maintained a threefold elevation throughout the captivity period, while body mass and T gradually decreased with time in captivity. BKA was lower at day 30, versus days 7 and 60, while peritoneal cell phagocytic efficiency decreased after day 30, remaining low at day 60. Moreover, phagocytosis efficiency was positively associated with T and body condition, suggesting that the effects of chronic stress on reproductive potential and immune response might be associated with the state of energetic reserves.

Melatoninergic System in Parkinson's Disease: From Neuroprotection to the Management of Motor and Nonmotor Symptoms.

Melatonin is synthesized by several tissues besides the pineal gland, and beyond its regulatory effects in light-dark cycle, melatonin is a hormone with neuroprotective, anti-inflammatory, and antioxidant properties. Melatonin acts as a free-radical scavenger, reducing reactive species and improving mitochondrial homeostasis. Melatonin also regulates the expression of neurotrophins that are involved in the survival of dopaminergic neurons and reduces α-synuclein aggregation, thus protecting the dopaminergic system against damage. The unbalance of pineal melatonin synthesis can predispose the organism to inflammatory and neurodegenerative diseases such as Parkinson's disease (PD). The aim of this review is to summarize the knowledge about the potential role of the melatoninergic system in the pathogenesis and treatment of PD. The literature reviewed here indicates that PD is associated with impaired brain expression of melatonin and its receptors MT1 and MT2. Exogenous melatonin treatment presented an outstanding neuroprotective effect in animal models of PD induced by different toxins, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat, and maneb. Despite the neuroprotective effects and the improvement of motor impairments, melatonin also presents the potential to improve nonmotor symptoms commonly experienced by PD patients such as sleep and anxiety disorders, depression, and memory dysfunction.

The concept of the immune-pineal axis tested in patients undergoing an abdominal hysterectomy.

OBJECTIVE: Activation of the immune-pineal axis induces a transient reduction in nocturnal melatonin in the plasma during the proinflammatory phase of an innate immune response to allow the proper migration of leukocytes to the lesion site. This transient reduction should be regulated by inflammatory mediators, which are responsible for the fine-tuning of the process. In the present study, we measured the pre- and postoperative serum concentrations of melatonin, tumor necrosis factor (TNF) and cortisol in women who underwent an elective hysterectomy and correlated the variation in melatonin with postoperative pain. METHODS: We evaluated 12 women who had an abdominal hysterectomy. Blood was collected at 10.00 and 22.00 h 1 week and 1 day before the surgery, on the 1st and 2nd days after the surgery and at 22.00 h on the day of the surgery. RESULTS: On the night after the surgery, there was no melatonin detected at 22.00 h. High TNF levels were accompanied by a lower nocturnal melatonin output, higher postoperative pain according to a visual analog scale and the request of higher doses of analgesics. In addition, low cortisol levels were accompanied by a lower nocturnal melatonin output. CONCLUSION: Our results confirm that the same antagonistic pattern between TNF and glucocorticoids observed in cultured pineal glands also occurs in humans. This integrative pattern suggests that the cross talk between the immune and endocrine system orchestrates longitudinal changes in pineal activity, reinforcing the hypothesis of an immune-pineal axis.

Endothelial cell adhesiveness is a function of environmental lighting and melatonin level.

The endothelial layer regulates the traffic of cells and substances between the blood and tissues and plays a central role in the mounting of an inflammatory response. We have recently shown that inhibition of the nocturnal melatonin surge during the mounting of an inflammatory response primes endothelial cells to a highly reactive state, increasing the expression of adhesion molecules and inducible nitric oxide synthase (iNOS) as well as the in vitro adherence of leukocytes. Here, we investigated whether physiological variations in the plasma melatonin levels owing to the light/dark environmental cycle could also prime the reactive state of endothelial cells. Cultured endothelial cells (16-20 days) obtained from rats killed during the daytime adhere more neutrophils, expressed more adhesion molecules and iNOS, and had a higher content of the transcription factor nuclear factor kappa B (NF-kB) translocated to the nuclei. We also evaluated the expression of 84 genes (using real-time PCR array) related to the innate inflammatory response and observed a higher expression of 19 genes in cultures obtained during the daytime. In addition, the only gene that was highly expressed in cells obtained from rats killed during nighttime was one that encodes a protein that negatively modulates inflammatory response. In conclusion, the daily rhythm of melatonin also primes the ability of endothelial cells to adhere to neutrophils. This new approach for evaluating the influence of the donor on cells maintained in culture should have applications for the standardization of cell banks.

Daily rhythm of salivary IL-1ß, cortisol and melatonin in day and night workers.

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.

Long-lasting priming of endothelial cells by plasma melatonin levels.

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.

TLR4 and CD14 receptors expressed in rat pineal gland trigger NFKB pathway.

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.