Antioxidant activity of glucosamine and its effects on ROS production, Nrf2, and O-GlcNAc expression in HMEC-1 cells.

Glucosamine (GlcN) is the most used supplement for osteoarthritis treatment. In vitro studies have related GlcN to beneficial and detrimental effects on health. The aim of this study was to evaluate the effects of O-linked-N-acetylglucosaminylation (O-GlcNAc) on GlcN-induced ROS production and Nrf2 expression in human dermal microvascular endothelial cells-1 (HMEC-1) and to evaluate the antioxidant capacity of GlcN compared to well-known antioxidants. For this, we evaluate the antioxidant capacity by in vitro assays. Besides, the GlcN (5-20 mM) effects on cell viability, reactive oxygen species (ROS) production, O-GlcNAc, and nuclear factor erythroid-2-related factor 2 (Nrf2) expression with and without the O-GlcNAc inhibitor OSMI-1 (10 µM) in HMEC-1 were evaluated. GlcN showed high inhibitory concentration (low scavenging activity) against superoxide (O2•─, IC20 = 47.67 mM), 2,2-diphenyl-1-picrylhydrazyl (DPPH•, IC50 = 21.32 mM), and hydroxyl (HO•, IC50 = 14.04 mM) radicals without scavenging activity against hydrogen peroxide (H2O2) and low antioxidant capacity determined by oxygen radical absorbance capacity (ORAC, 0.001 mM Trolox equivalent) and ferric reducing antioxidant power (FRAP, 0.046 mM Trolox equivalent). In cell culture, GlcN (20 mM) reduced cell viability up to 26 % and induced an increase in ROS production (up to 70 %), O-GlcNAc (4-fold-higher vs. control), and Nrf2 expression (56 %), which were prevented by OSMI-1. These data suggest an association between O-GlcNAc, ROS production, and Nrf2 expression in HMEC-1 cells stimulated with GlcN.

Motor imagery EEG signal classification with a multivariate time series approach.

BACKGROUND: Electroencephalogram (EEG) signals record electrical activity on the scalp. Measured signals, especially EEG motor imagery signals, are often inconsistent or distorted, which compromises their classification accuracy. Achieving a reliable classification of motor imagery EEG signals opens the door to possibilities such as the assessment of consciousness, brain computer interfaces or diagnostic tools. We seek a method that works with a reduced number of variables, in order to avoid overfitting and to improve interpretability. This work aims to enhance EEG signal classification accuracy by using methods based on time series analysis. Previous work on this line, usually took a univariate approach, thus losing the possibility to take advantage of the correlation information existing within the time series provided by the different electrodes. To overcome this problem, we propose a multivariate approach that can fully capture the relationships among the different time series included in the EEG data. To perform the multivariate time series analysis, we use a multi-resolution analysis approach based on the discrete wavelet transform, together with a stepwise discriminant that selects the most discriminant variables provided by the discrete wavelet transform analysis RESULTS: Applying this methodology to EEG data to differentiate between the motor imagery tasks of moving either hands or feet has yielded very good classification results, achieving in some cases up to 100% of accuracy for this 2-class pre-processed dataset. Besides, the fact that these results were achieved using a reduced number of variables (55 out of 22,176) can shed light on the relevance and impact of those variables. CONCLUSIONS: This work has a potentially large impact, as it enables classification of EEG data based on multivariate time series analysis in an interpretable way with high accuracy. The method allows a model with a reduced number of features, facilitating its interpretability and improving overfitting. Future work will extend the application of this classification method to help in diagnosis procedures for detecting brain pathologies and for its use in brain computer interfaces. In addition, the results presented here suggest that this method could be applied to other fields for the successful analysis of multivariate temporal data.

Transcriptional activity and splicing factors are preserved during physiological apoptosis.

Apoptosis is the best-known programmed cell death that maintains tissue homeostasis in eukaryotic cells. The morphological characteristics include nuclear and cytoplasmic contraction and cytoplasmic blebbing, its biochemical hallmarks include caspase protease activity and DNA fragmentation. In rat ovaries, cell death is a normal process that occurs throughout the organism's life. Granulosa cells, the more abundant cell type forming the ovarian follicles, are eliminated via different routes of cell death. Most granulosa cells are eliminated through apoptotic cell death. In this work, we analyzed the behavior of nuclear components throughout the apoptotic process and determined how they are regionalized and conserved during follicular atresia in rat ovaries. Apoptosis was detected based on caspase-3 activity and DNA fragmentation using the TUNEL technique. We identified the transcription markers H3ac and RNA Pol II, and splicing factor SC35 by immunodetection. The nucleolar components were analyzed via light microscopy and transmission electron microscopy through immunodetection of the proteins nucleolin and nucleophosmin-1. The nuclear ultrastructure was analyzed using standard contrast and preferential ribonucleoprotein contrast. Our results demonstrate that during the progression of apoptosis, chromatin is remodeled to constitute apoptotic bodies; transcription and spliceosome elements are reorganized along with the nucleolar components. Additionally, the splicing and transcription factors are segregated into specific territories inside the apoptotic bodies, suggesting that transcriptional elements are reorganized during the apoptotic process. Our results indicate that apoptotic bodies not only are compacted, and chromatin degraded but all the nuclear components are progressively reorganized during cell elimination; moreover, the transcriptional components are preserved.

Iodine deficiency disturbs the metabolic profile and elemental composition of human breast milk.

Human breast milk (HBM) has a beneficial impact on health programming, growth and neurodevelopment of newborns.Increase in iodine intake is recommended for pregnant women in order to produce enough thyroid hormones to meet foetal requirements.In this work, a combined analytical multiplatform based on gas chromatography coupled to mass spectrometry and ultra-high performance liquid chromatography coupled toquadrupole-time-of-flight mass spectrometryhas been appliedinthe first metabolomic study of HBM ofiodine-deficientwomen. In addition, the elemental composition of HBM has been determined by inductively coupled plasma triple quadrupole mass spectrometry. Remarkably,31 metaboliteswith important biological roles(e.g. glycerophospholipids for neurodevelopment)were seentobe alteredin the HBM of iodine-deficient women. The main metabolic pathwaysalteredinclude lipid metabolism, amino acid cycle, the tricarboxylic acid cycle and glycolysis.Additionally, the concentration of selenium, zinc and copperwere seento be significantlylowerin HBM of iodine-deficient women.

A novel HPLC column switching method coupled to ICP-MS/QTOF for the first determination of selenoprotein P (SELENOP) in human breast milk.

In this work, we describe for the first time the presence of selenoprotein P in human breast milk. To this end, a novel analytical method has been developed based on a two-dimensional column switching system, which consisted of three size exclusion columns and one affinity column coupled to inductively coupled plasma mass spectrometry (ICP-MS). The method combines the accurate quantification of selenoproteins and selenometabolites by species unspecific isotopic dilution ICP-MS, with unequivocal identification by quadrupole-time-of-flight mass spectrometry. Several selenopeptides, which contain the amino acid selenocysteine (U, SeCys), were identified after tryptic digestion followed by their separation. The results reveal that the relative selenium concentration in colostrum follows the order: glutathione peroxidase (GPX) ≈ selenoprotein P (SELENOP) > selenocystamine (SeCA) > other selenometabolites (SeMB), in contrast with previously published papers (GPX > SeCA > selenocystine > selenomethionine). A mean concentration of 20.1 ± 1.0 ng Se g-1 as SELENOP (1.45 µg SELENOP/g) was determined in colostrum (31% of total selenium).

Novel mechanisms for the metabolic control of puberty: implications for pubertal alterations in early-onset obesity and malnutrition.

Puberty is driven by sophisticated neuroendocrine networks that timely activate the brain centers governing the reproductive axis. The timing of puberty is genetically determined; yet, puberty is also sensitive to numerous internal and external cues, among which metabolic/nutritional signals are especially prominent. Compelling epidemiological evidence suggests that alterations of the age of puberty are becoming more frequent; the underlying mechanisms remain largely unknown, but the escalating prevalence of obesity and other metabolic/feeding disorders is possibly a major contributing factor. This phenomenon may have clinical implications, since alterations in pubertal timing have been associated to adverse health outcomes, including higher risk of earlier all-cause mortality. This urges for a better understanding of the neurohormonal basis of normal puberty and its deviations. Compelling evidence has recently documented the master role of hypothalamic neurons producing kisspeptins, encoded by Kiss1, in the neuroendocrine pathways controlling puberty. Kiss1 neurons seemingly participate in transmitting the regulatory actions of metabolic cues on pubertal maturation. Key cellular metabolic sensors, as the mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK) and the fuel-sensing deacetylase, SIRT1, have been recently shown to participate also in the metabolic modulation of puberty. Recently, we have documented that AMPK and SIRT1 operate as major molecular effectors for the metabolic control of Kiss1 neurons and, thereby, puberty onset. Alterations of these molecular pathways may contribute to the perturbation of pubertal timing linked to conditions of metabolic stress in humans, such as subnutrition or obesity and might become druggable targets for better management of pubertal disorders.

SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression.

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

Iodine status and thyroid function among Spanish schoolchildren aged 6-7 years: the Tirokid study.

I deficiency is still a worldwide public health problem, with children being especially vulnerable. No nationwide study had been conducted to assess the I status of Spanish children, and thus an observational, multicentre and cross-sectional study was conducted in Spain to assess the I status and thyroid function in schoolchildren aged 6-7 years. The median urinary I (UI) and thyroid-stimulating hormone (TSH) levels in whole blood were used to assess the I status and thyroid function, respectively. A FFQ was used to determine the consumption of I-rich foods. A total of 1981 schoolchildren (52 % male) were included. The median UI was 173 µg/l, and 17·9 % of children showed UI<100 µg/l. The median UI was higher in males (180·8 v. 153·6 µg/l; P<0·001). Iodised salt (IS) intake at home was 69·8 %. IS consumption and intakes of ≥2 glasses of milk or 1 cup of yogurt/d were associated with significantly higher median UI. Median TSH was 0·90 mU/l and was higher in females (0·98 v. 0·83; P<0·001). In total, 0·5 % of children had known hypothyroidism (derived from the questionnaire) and 7·6 % had TSH levels above reference values. Median TSH was higher in schoolchildren with family history of hypothyroidism. I intake was adequate in Spanish schoolchildren. However, no correlation was found between TSH and median UI in any geographical area. The prevalence of TSH above reference values was high and its association with thyroid autoimmunity should be determined. Further assessment of thyroid autoimmunity in Spanish schoolchildren is desirable.

Dolor fantasma en niños y jóvenes amputados adquiridos: prevalencia y características clínicas / Phantom pain in children and teenagers with acquired amputations: prevalence an clinical features

Introducción: El dolor fantasma es una condición frecuente en pacientes amputados lo que genera discapacidad importante. En población adulta se estima una prevalencia entre 49-82 por ciento. Existe escasa evidencia de la incidencia y prevalencia en población de niños y adolescentes. Objetivos: Estimar la incidencia y la prevalencia del dolor fantasma en la población de amputados adquiridos del Instituto Teletón (IT) de Santiago de 10 años y más; caracterizar a esta población y asociar distintos factores clínicos y demográficos con la presencia de este dolor. Pacientes y Métodos: Estudio descriptivo de incidencia y prevalencia basado en la revisión de fichas clínicas para la obtención de datos demográficos, clínicos y evaluación de registro de dolor fantasma. Se aplicó encuesta telefónica a pacientes de 10 años o más, con diagnóstico de una o más amputación/es adquirida/s, que se atiendan o hayan sido atendido en IT Santiago hasta el año 2013. Resultados: La incidencia de dolor fantasma en la población estudiada es de 11 por 100 personas/año y prevalencia de 62 por ciento. Se encontraron asociaciones estadísticamente significativas, entre la presencia de dolor fantasma y el tiempo transcurrido desde la amputación (a mayor tiempo transcurrido, menor dolor) y edad de la amputación (a mayor edad en que se realizó la amputación mayor dolor). Conclusión: El dolor fantasma es un fenómeno frecuente en pacientes amputados adquiridos de 10 años y más atendidos en el IT de Santiago con una prevalencia del 62 por ciento.

Introduction: Phantom pain is a common condition in amputated patients generating significant disability. Prevalence among adult population is estimated at 49-82 percent. There is little evidence of the incidence and prevalence in child and adolescent population. Objectives: To estimate the incidence and prevalence of phantom pain in amputees aged 10 years and older of the Telethon Institute of Santiago; characterize this population and associate different clinical and demographic factors with the presence of phantom pain. Patients and Methods: Study incidence and prevalence based on a review of medical records to obtain demographic and clinical data. In addition a telephone survey was made to patients 10 years or older diagnosed with one or more acquired amputations who are treated or have been treated at Telethon Institute of Santiago until 2013. Results: The incidence of phantom pain in the study population is 11 per 100 persons/year and prevalence is 62 percent. Statistically significant associations were found between the presence of phantom pain variables and time since amputation (the longer the time elapsed, less pain) and age of amputation (the older the age at which major amputation was performed more pain). Conclusion: Phantom pain is a common phenomenon in patients with acquired amputation aged 10 years and older treated at the Telethon Institute of Santiago prevalence being 62 percent.

Effect of the ketamine/xylazine anesthetic on the auditory brainstem response of adult gerbils

The auditory brainstem response (ABR) is a test widely used to assess the integrity of the brain stem. Although it is considered to be an auditory-evoked potential that is influenced by the physical characteristics of the stimulus, such as rate, polarity and type of stimulus, it may also be influenced by the change in several parameters. The use of anesthetics may adversely influence the value of the ABR wave latency. One of the anesthetics used for e ABR assessment, especially in animal research, is the ketamine/xylazine combination. Our objective was to determine the influence of the ketamine/xylazine anesthetic on the ABR latency values in adult gerbils. The ABRs of 12 adult gerbils injected with the anesthetic were collected on three consecutive days, or a total of six collections, namely: pre-collection and A, B, C, D, and E collections. Before each collection the gerbil was injected with a dose of ketamine (100 mg/kg)/xylazine (4 mg/kg). For the capture of the ABR, 2000 click stimuli were used with rarefaction polarity and 13 stimuli per second, 80 dBnHL intensity and in-ear phones. A statistically significant difference was observed in the latency of the V wave in the ABR of gerbils in the C and D collections compared to the pre-, A and E collections, and no difference was observed between the pre-, A, B, and E collections. We conclude that the use of ketamine/xylazine increases the latency of the V wave of the ABR after several doses injected into adult gerbils; thus clinicians should consider the use of this substance in the assessment of ABR.

Effect of the ketamine/xylazine anesthetic on the auditory brainstem response of adult gerbils.

The auditory brainstem response (ABR) is a test widely used to assess the integrity of the brain stem. Although it is considered to be an auditory-evoked potential that is influenced by the physical characteristics of the stimulus, such as rate, polarity and type of stimulus, it may also be influenced by the change in several parameters. The use of anesthetics may adversely influence the value of the ABR wave latency. One of the anesthetics used for e ABR assessment, especially in animal research, is the ketamine/xylazine combination. Our objective was to determine the influence of the ketamine/xylazine anesthetic on the ABR latency values in adult gerbils. The ABRs of 12 adult gerbils injected with the anesthetic were collected on three consecutive days, or a total of six collections, namely: pre-collection and A, B, C, D, and E collections. Before each collection the gerbil was injected with a dose of ketamine (100 mg/kg)/xylazine (4 mg/kg). For the capture of the ABR, 2000 click stimuli were used with rarefaction polarity and 13 stimuli per second, 80 dBnHL intensity and in-ear phones. A statistically significant difference was observed in the latency of the V wave in the ABR of gerbils in the C and D collections compared to the pre-, A and E collections, and no difference was observed between the pre-, A, B, and E collections. We conclude that the use of ketamine/xylazine increases the latency of the V wave of the ABR after several doses injected into adult gerbils; thus clinicians should consider the use of this substance in the assessment of ABR.

Histamine is required during neural stem cell proliferation to increase neuron differentiation.

Histamine in the adult central nervous system (CNS) acts as a neurotransmitter. This amine is one of the first neurotransmitters to appear during development reaching its maximum concentration simultaneously with neuron differentiation peak. This suggests that HA plays an important role in neurogenesis. We have previously shown that HA is able to increase neuronal differentiation of neural stem cells (NSCs) in vitro, by activating the histamine type 1 receptor. However the mechanism(s) by which HA has a neurogenic effect on NSCs has not been explored. Here we explore how HA is able to increase neuron phenotype. Cortex neuroepithelium progenitors were cultured and at passage two treatments with 100 µM HA were given during cell proliferation and differentiation or only during differentiation. Immunocytochemistry was performed on differentiated cultures to detect mature neurons. To explore the expression of certain important transcriptional factors involved on asymmetric cell division and commitment, RT-PCR and qRT-PCR were performed. Results indicate that HA is required during cell proliferation in order to increase neuron differentiation and suggest that this amine increases neuron commitment during the proliferative phase probably by rising prospero1 and neurogenin1 expression.

Iodine intake in the adult population. Di@bet.es study.

BACKGROUND & AIMS: To date no nation-wide study has yet been undertaken in Spain to estimate the iodine deficiency. The aim was to evaluate iodine intake and its conditioning factors in a representative sample of the whole adult population. METHODS: The Di@bet.es Study is a national, cross-sectional, population-based survey conducted in 2009-2010 in Spain. RESULTS: The median urinary iodine (UI) was 117.2 µg/L. Iodized salt (IS) was consumed by 43.9% of the population. The median UI in those who consumed IS and in those who did not consume IS was 131.1 and 110.8 µg/L respectively (p<0.0001). The likelihood of having UI levels above 100 µg/L was significantly associated with the intake of IS (OR=1.47) and milk at least once a day (OR=1.22). Within each individual autonomous communities, the median UI levels in those who consumed IS correlated significantly with the median levels of those who did not consume IS (r=0.76, p=0.001). CONCLUSIONS: Though strictly speaking, Spain should be considered within the category of a country having an adequate iodine intake, the current value is too close to the cut point and does not guarantee that those groups with a greater need for iodine will have the required intake of iodine.

Unilateral variation in the origin of the inferior alveolar and buccal arteries: a case report / Variación unilateral en el origen de las arterias alveolar inferior y bucal: reporte de un caso

The maxillary artery (MA) is one of the terminal branches of the external carotid artery (ECA) and is located in the infratemporal fossa (IF). Some of the branches in this region are the inferior alveolar artery (IAA) and the buccal artery (BA), both descending branches. Here, we report an unusual unilateral origin of the IAA and the BA from a common trunk directly from the ECA. We conducted a routine dissection of both IF in a 54-year-old hispanic male cadaver. Fixed with Universidad de los Andes® conservative solution and red latex for vascular filling. On each side, the MA is observed superficially located over the lateral pterygoid muscle. On the right side, the IAA and the BA originate from a common trunk from the ECA approximately 5 mm prior to the bifurcation into their terminal branches. On the left side, the IAA originates from the MA that is immediately next to its origin, making a common trunk with the pterygoid branches. Knowing the morphology of the MA and its branches at the IF is important for oral and maxillofacial surgery procedures; and any variation in the origin or course of these arteries may result in the patient's increased morbidity during some invasive procedure in the area.

La arteria maxilar (AM) es una rama terminal de la arteria carótida externa (ACE), y se ubica en la región infratemporal (RI). Algunas de sus ramas en esta región son la arteria alveolar inferior (AAI) y la arteria bucal (AB), ambas ramas descendentes. En este trabajo informamos de un inusual origen unilateral de la AAI y de la AB a partir de un tronco común desde la ACE. Se realizó una disección de rutina de ambas regiones infratemporales en un cadáver de 54 años, sexo masculino, caucásico. Fijado con solución conservadora Universidad de los Andes® y repleción vascular con látex rojo. A cada lado, se observa la AM en ubicación superficial sobre el músculo pterigoideo lateral. Al lado derecho, la AAI y la AB se originan de un tronco común desde la ACE aproximadamente 5 mm antes de la bifurcación en sus ramas terminales. Al lado izquierdo la AAI se origina de la AM inmediato a su origen, formando un tronco común con los ramos pterigoideos. El conocimiento de la morfología de la AM y de sus ramas en la RI es de importancia en procedimientos odontológicos, de cirugía oral y maxilofacial. Por lo que cualquier variación en el origen o trayecto de estas arterias puede predisponer a un paciente a una mayor morbilidad durante algún procedimiento invasivo en la zona.

Tumor odontogénico adenomatoide en maxilar: reporte de un caso y revisiónde la literatura / Adenomatoid odontogenic tumor in maxilla: a report case and review of the literature

El tumor odontogénico adenomatoide es una neoplasia benigna según la nueva clasificación del 2005 sobre tumores odontogénicos. Es una lesión muchas veces de crecimiento lento pero progresivo y no invasivo. Este reporte describe una paciente de 17 años, sexo femenino, con un tumor odontogénico adenomatoide del subtipo folicular en la región maxilar anterior comprometiendo al canino maxilar izquierdo. Además se realiza una revisión para los profesionales de la salud sobre aspectos diagnósticos y manejo según los hallazgos más recientes en esta patología.

Adenomatoid odontogenic tumor is a benign neoplasm in the new classification of odontogenic tumors of 2005. This lesion is often slow growing but progressive and noninvasive. This report describes a 17-year old female patient with an adenomatoid odontogenic tumor follicular subtype in the anterior maxillary region compromising the left maxillary canine. This article also provides a refresher for the health professionals on its diagnosis and management according to recent findings in this pathology.

PPARγ expression is increased in systemic lupus erythematosus patients and represses CD40/CD40L signaling pathway.

Systemic lupus erythematosus (SLE) is a heterogeneous disease involving several immune cell types and pro-inflammatory signals, including the one triggered by binding of CD40L to the receptor CD40. Peroxisome-proliferator activated receptor gamma (PPARγ) is a transcription factor with anti-inflammatory properties. Here we investigated whether CD40 and PPARγ could exert opposite effects in the immune response and the possible implications for SLE. Increased PPARγ mRNA levels were detected by real-time PCR in patients with active SLE, compared to patients with inactive SLE PPARγ/GAPDH mRNA = 2.21 ± 0.49 vs. 0.57 ± 0.14, respectively (p < 0.05) or patients with infectious diseases and healthy subjects (p < 0.05). This finding was independent of the corticosteroid therapy. We further explored these observations in human THP1 and in SLE patient-derived macrophages, where activation of CD40 by CD40L promoted augmented PPARγ gene transcription compared to non-stimulated cells (PPARγ/GAPDH mRNA = 1.14 ± 0.38 vs. 0.14 ± 0.01, respectively; p < 0.05). This phenomenon occurred specifically upon CD40 activation, since lipopolysaccharide treatment did not induce a similar response. In addition, increased activity of PPARγ was also detected after CD40 activation, since higher PPARγ-dependent transcription of CD36 transcription was observed. Furthermore, CD40L-stimulated transcription of CD80 gene was elevated in cells treated with PPARγ-specific small interfering RNA (small interfering RNA, siRNA) compared to cells treated with CD40L alone (CD80/GAPDH mRNA = 0.11 ± 0.04 vs. 0.05 ± 0.02, respectively; p < 0.05), suggesting a regulatory role for PPARγ on the CD40/CD40L pathway. Altogether, our findings outline a novel mechanism through which PPARγ regulates the inflammatory signal initiated by activation of CD40, with important implications for the understanding of immunological mechanisms underlying SLE and the development of new treatment strategies.

Immune cells and oxidative stress in the endotoxin tolerance mouse model

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Immune cells and oxidative stress in the endotoxin tolerance mouse model.

Sepsis is a systemic inflammatory response that can lead to tissue damage and death. In order to increase our understanding of sepsis, experimental models are needed that produce relevant immune and inflammatory responses during a septic event. We describe a lipopolysaccharide tolerance mouse model to characterize the cellular and molecular alterations of immune cells during sepsis. The model presents a typical lipopolysaccharide tolerance pattern in which tolerance is related to decreased production and secretion of cytokines after a subsequent exposure to a lethal dose of lipopolysaccharide. The initial lipopolysaccharide exposure also altered the expression patterns of cytokines and was followed by an 8- and a 1.5-fold increase in the T helper 1 and 2 cell subpopulations. Behavioral data indicate a decrease in spontaneous activity and an increase in body temperature following exposure to lipopolysaccharide. In contrast, tolerant animals maintained production of reactive oxygen species and nitric oxide when terminally challenged by cecal ligation and puncture (CLP). Survival study after CLP showed protection in tolerant compared to naive animals. Spleen mass increased in tolerant animals followed by increases of B lymphocytes and subpopulation Th1 cells. An increase in the number of stem cells was found in spleen and bone marrow. We also showed that administration of spleen or bone marrow cells from tolerant to naive animals transfers the acquired resistance status. In conclusion, lipopolysaccharide tolerance is a natural reprogramming of the immune system that increases the number of immune cells, particularly T helper 1 cells, and does not reduce oxidative stress.

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and “re-couples” NOS function.

Purine nucleotides reduce superoxide production by nitric oxide synthase in a murine sepsis model.

Sepsis involves a systemic inflammatory response of multiple endogenous mediators, resulting in many of the injurious and sometimes fatal physiological symptoms of the disease. This systemic activation leads to a compromised vascular response and endothelial dysfunction. Purine nucleotides interact with purinoceptors and initiate a variety of physiological processes that play an important role in maintaining cardiovascular function. The purpose of the present study was to investigate the effects of ATP on vascular function in a lipopolysaccharide (LPS) model of sepsis. LPS induced a significant increase in aortic superoxide production 16 h after injection. Addition of ATP to the organ bath incubation solution reduced superoxide production by the aortas of endotoxemic animals. Reactive Blue, an antagonist of the P2Y receptor, blocked the effect of ATP on superoxide production, and the nonselective P2Y agonist MeSATP inhibited superoxide production. Nitric oxide synthase (NOS) inhibition by L-NAME blocked vascular relaxation and reduced superoxide production in LPS-treated animals. In the presence of L-NAME there was no ATP effect on superoxide production. A vascular reactivity study showed that ATP increased maximal relaxation in LPS-treated animals compared to controls. The presence of ATP induced increases in Akt and endothelial NOS phosphorylated proteins in the aorta of septic animals. ATP reduces superoxide release resulting in an improved vasorelaxant response. Sepsis may uncouple NOS to produce superoxide. We showed that ATP through Akt pathway phosphorylated endothelial NOS and "re-couples" NOS function.