Assessment of diesel exhaust particulate exposure and surface characteristics in association with levels of oxidative stress biomarkers

Exposure to PM10 and PM2.5 (particulate matter with aerodynamic diameter smaller than 10 μm and 2.5 μm, respectively) is associated with a range of adverse health effects, including cancer, pulmonary and cardiovascular diseases. Surface characteristics (chemical reactivity, surface area) are considered of prime importance to understand the mechanisms which lead to harmful effects. A hypothetical mechanism to explain these adverse effects is the ability of components (organics, metal ions) adsorbed on these particles to generate Reactive Oxygen Species (ROS), and thereby to cause oxidative stress in biological systems (Donaldson et al., 2003). ROS can attack almost any cellular structure, like DNA or cellular membrane, leading to the formation of a wide variety of degradation products which can be used as a biomarker of oxidative stress. The aim of the present research project is to test whether there is a correlation between the exposure to Diesel Exhaust Particulate (DEP) and the oxidative stress status. For that purpose, a survey has been conducted in real occupational situations where workers were exposed to DEP (bus depots). Different exposure variables have been considered: - particulate number, size distribution and surface area (SMPS); - particulate mass - PM2.5 and PM4 (gravimetry); - elemental and organic carbon (coulometry); - total adsorbed heavy metals - iron, copper, manganese (atomic adsorption); - surface functional groups present on aerosols (Knudsen flow reactor). Several biomarkers of oxidative stress (8-hydroxy-2'-deoxyguanosine and several aldehydes) have been determined either in urine or serum of volunteers. Results obtained during the sampling campaign in several bus depots indicated that the occupational exposure to particulates in these places was rather low (40-50 μg/m3 for PM4). Bimodal size distributions were generally observed (5 μm and <1 μm). Surface characteristics of PM4 varied strongly, depending on the bus depot. They were usually characterized by high carbonyl and low acidic sites content. Among the different biomarkers which have been analyzed within the framework of this study, mean urinary levels of 8-hydroxy-2'-deoxyguanosine increased significantly (p<0.05) during two consecutive days of exposure for non-smoker workers. On the other hand, no statistically significant differences were observed for serum levels of hexanal, nonanal and 4- hydroxy-nonenal (p>0.05). Biomarkers levels will be compared to exposure variables to gain a better understanding of the relation between the particulate characteristics and the formation of ROS by-products. This project is financed by the Swiss State Secretariat for Education and Research. It is conducted within the framework of the COST Action 633 "Particulate Matter - Properties Related to Health Effects".

Distribution of plasma levels of adiponectin and leptin in an adult Caucasian population.

OBJECTIVES: Little is known regarding the distribution and the determinants of leptin and adiponectin levels in the general population. DESIGN: Cross-sectional study. PATIENTS: Women (3004) and men (2552) aged 35-74 living in Lausanne, Switzerland. MEASUREMENTS: Plasma levels of leptin and adiponectin (ELISA measurement). RESULTS: Women had higher leptin and adiponectin levels than men. In both genders, leptin and adiponectin levels increased with age. After adjusting for fat mass, leptin levels were significantly and negatively associated with age in women: 18.1 +/- 0.3, 17.1 +/- 0.3, 16.7 +/- 0.3 and 15.5 +/- 0.4 ng/ml (adjusted mean +/- SE) for age groups [35-44], [45-54], [55-64] and [65-75], respectively, P < 0.001. A similar but nonsignificant trend was also found in men. Conversely, the age-related increase of adiponectin was unrelated to body fat in both genders. Post-menopausal women had higher leptin and adiponectin levels than premenopausal women, independently of hormone replacement therapy. Although body fat mass was associated with leptin and adiponectin, the associations were stronger with body mass index (BMI), waist and hip in both genders. Finally, after adjusting for age and anthropometry, no relationships were found between leptin or adiponectin levels with alcohol, caffeine consumption and physical activity, whereas smoking and diabetes decreased leptin and adiponectin levels in women only. CONCLUSIONS: The age-related increase in leptin levels is attributable to changes in fat mass in women and probably also in men. Leptin and adiponectin levels are more related to BMI than to body fat mass. The effects of smoking and diabetes appear to be gender-specific.

Simultaneous determination of human plasma levels of citalopram, paroxetine, sertraline, and their metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry method is presented which allows the simultaneous determination of the plasma concentrations of the selective serotonin reuptake inhibitors citalopram, paroxetine, sertraline, and their pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline) after derivatization with the reagent N-methyl-bis(trifluoroacetamide). No interferences from endogenous compounds are observed following the extraction of plasma samples from six different human subjects. The standard curves are linear over a working range of 10-500 ng/mL for citalopram, 10-300 ng/mL for desmethylcitalopram, 5-60 ng/mL for didesmethylcitalopram, 20-400 ng/mL for sertraline and desmethylsertraline, and 10-200 ng/mL for paroxetine. Recoveries measured at three concentrations range from 81 to 118% for the tertiary amines (citalopram and the internal standard methylmaprotiline), 73 to 95% for the secondary amines (desmethylcitalopram, paroxetine and sertraline), and 39 to 66% for the primary amines (didesmethylcitalopram and desmethylsertraline). Intra- and interday coefficients of variation determined at three concentrations range from 3 to 11% for citalopram and its metabolites, 4 to 15% for paroxetine, and 5 to 13% for sertraline and desmethylsertraline. The limits of quantitation of the method are 2 ng/mL for citalopram and paroxetine, 1 ng/mL for sertraline, and 0.5 ng/mL for desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline. No interferences are noted from 20 other psychotropic drugs. This sensitive and specific method can be used for single-dose pharmacokinetics. It is also useful for therapeutic drug monitoring of these three drugs and could possibly be adapted for the quantitation of the two other selective serotonin reuptake inhibitors on the market, namely fluoxetine and fluvoxamine.

The effect of 4-chlororesorcinol on the endogenous levels of IAA, ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3-4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.

Behavioral changes in brain-injured critical care adults with different levels of consciousness during nociceptive stimulation: an observational study.

PURPOSE: The primary objective of this study was to describe the frequency of behaviors observed during rest, a non-nociceptive procedure, and a nociceptive procedure in brain-injured intensive care unit (ICU) patients with different levels of consciousness (LOC). Second, it examined the inter-rater reliability and discriminant and concurrent validity of the behavioral checklist used. METHODS: The non-nociceptive procedure involved calling the patient and shaking his/her shoulder. The nociceptive procedure involved turning the patient. The frequency of behaviors was recorded using a behavioral checklist. RESULTS: Patients with absence of movement, or stereotyped flexion or extension responses to a nociceptive stimulus displayed more behaviors during turning (median 5.5, range 0-14) than patients with localized responses (median 4, range 0-10) or able to self-report their pain (median 4, range 0-10). Face flushing, clenched teeth, clenched fist, and tremor were more frequent in patients with absence of movement, or stereotyped responses to a nociceptive stimulus. The reliability of the checklist was supported by a high intra-class correlation coefficient (0.77-0.92), and the internal consistency was acceptable in all three groups (KR 20, 0.71-0.85). Discriminant validity was supported as significantly more behaviors were observed during nociceptive stimulation than at rest. Concurrent validity was confirmed as checklist scores were correlated to the patients' self-reports of pain (r s = 0.53; 95 % CI 0.21-0.75). CONCLUSION: Brain-injured patients reacted significantly more during a nociceptive stimulus and the number of observed behaviors was higher in patients with a stereotyped response.

Nestmate recognition and levels of aggression are not altered by changes in genetic diversity in a socially polymorphic ant

The ability to distinguish nestmates from foreign individuals is central to the functioning of insect societies. In ants, workers from multiple-queen colonies are often less aggressive than workers from single-queen ones. In line with this observation, it has been hypothesized that workers from multiple-queen colonies have less precise recognition abilities than workers from single-queen ones because their colonies contain genetically more diverse individuals, which results in a broader template of recognition cues. Here, we assessed the impact of social structure ( queen number) variation on nestmate recognition and aggression in a large population of the socially polymorphic ant Formica selysi. We staged unilateral aggression tests on the nest surface. Workers from single-and multiple-queen colonies had good nestmate recognition ability and did not differ significantly in their level of aggression towards foreign, immobilized workers ( cue-bearers). In particular, workers from multiple-queen colonies efficiently recognized non-nestmates despite the higher genetic diversity in their colony. Cue-bearers from single- and multiple-queen colonies elicited similar reactions. However, the level of aggression was higher between than within social forms, suggesting that workers detect a signal that is specific to the colony social structure. Finally, the level of aggression was not correlated with the genetic distance between colonies. Overall, we found no evidence for the hypothesis that the presence of multiple breeders in the same colony decreases recognition abilities and found no simple relationship between genetic diversity and aggression level. (c) 2007 The Association for the Study of Animal Behaviou

Attenuated Levels of Hippocampal Connexin 43 and its Phosphorylation Correlate with Antidepressant- and Anxiolytic-Like Activities in Mice.

Clinical and preclinical studies have implicated glial anomalies in major depression. Conversely, evidence suggests that the activity of antidepressant drugs is based, at least in part, on their ability to stimulate density and/or activity of astrocytes, a major glial cell population. Despite this recent evidence, little is known about the mechanism(s) by which astrocytes regulate emotionality. Glial cells communicate with each other through gap junction channels (GJCs), while they can also directly interact with neurons by releasing gliotransmitters in the extracellular compartment via an hemichannels (HCs)-dependent process. Both GJCs and HCs are formed by two main protein subunits: connexins (Cx) 30 and 43 (Cx30 and Cx43). Here we investigate the role of hippocampal Cx43 in the regulation of depression-like symptoms using genetic and pharmacological approaches. The first aim of this study was to evaluate the impact of the constitutive knock-down of Cx43 on a set of behaviors known to be affected in depression. Conversely, the expression of Cx43 was assessed in the hippocampus of mice subjected to prolonged corticosterone (CORT) exposure, given either alone or in combination with an antidepressant drug, the selective serotonin reuptake inhibitor fluoxetine. Our results indicate that the constitutive deficiency of Cx43 resulted in the expression of some characteristic hallmarks of antidepressant-/anxiolytic-like behavioral activities along with an improvement of cognitive performances. Moreover, in a new cohort of wild-type mice, we showed that CORT exposure elicited anxiety and depression-like abnormalities that were reversed by chronic administration of fluoxetine. Remarkably, CORT also increased hippocampal amounts of phosphorylated form of Cx43 whereas fluoxetine treatment normalized this parameter. From these results, we envision that antidepressant drugs may exert their therapeutic activity by decreasing the expression and/or activity of Cx43 resulting from a lower level of phosphorylation in the hippocampus.

Decreased Brain Levels of Vitamin B12 in Aging, Autism and Schizophrenia.

Many studies indicate a crucial role for the vitamin B12 and folate-dependent enzyme methionine synthase (MS) in brain development and function, but vitamin B12 status in the brain across the lifespan has not been previously investigated. Vitamin B12 (cobalamin, Cbl) exists in multiple forms, including methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl), serving as cofactors for MS and methylmalonylCoA mutase, respectively. We measured levels of five Cbl species in postmortem human frontal cortex of 43 control subjects, from 19 weeks of fetal development through 80 years of age, and 12 autistic and 9 schizophrenic subjects. Total Cbl was significantly lower in older control subjects (> 60 yrs of age), primarily reflecting a >10-fold age-dependent decline in the level of MeCbl. Levels of inactive cyanocobalamin (CNCbl) were remarkably higher in fetal brain samples. In both autistic and schizophrenic subjects MeCbl and AdoCbl levels were more than 3-fold lower than age-matched controls. In autistic subjects lower MeCbl was associated with decreased MS activity and elevated levels of its substrate homocysteine (HCY). Low levels of the antioxidant glutathione (GSH) have been linked to both autism and schizophrenia, and both total Cbl and MeCbl levels were decreased in glutamate-cysteine ligase modulatory subunit knockout (GCLM-KO) mice, which exhibit low GSH levels. Thus our findings reveal a previously unrecognized decrease in brain vitamin B12 status across the lifespan that may reflect an adaptation to increasing antioxidant demand, while accelerated deficits due to GSH deficiency may contribute to neurodevelopmental and neuropsychiatric disorders.

High expression levels of macrophage migration inhibitory factor sustain the innate immune responses of neonates.

The vulnerability to infection of newborns is associated with a limited ability to mount efficient immune responses. High concentrations of adenosine and prostaglandins in the fetal and neonatal circulation hamper the antimicrobial responses of newborn immune cells. However, the existence of mechanisms counterbalancing neonatal immunosuppression has not been investigated. Remarkably, circulating levels of macrophage migration inhibitory factor (MIF), a proinflammatory immunoregulatory cytokine expressed constitutively, were 10-fold higher in newborns than in children and adults. Newborn monocytes expressed high levels of MIF and released MIF upon stimulation with Escherichia coli and group B Streptococcus, the leading pathogens of early-onset neonatal sepsis. Inhibition of MIF activity or MIF expression reduced microbial product-induced phosphorylation of p38 and ERK1/2 mitogen-activated protein kinases and secretion of cytokines. Recombinant MIF used at newborn, but not adult, concentrations counterregulated adenosine and prostaglandin E2-mediated inhibition of ERK1/2 activation and TNF production in newborn monocytes exposed to E. coli. In agreement with the concept that once infection is established high levels of MIF are detrimental to the host, treatment with a small molecule inhibitor of MIF reduced systemic inflammatory response, bacterial proliferation, and mortality of septic newborn mice. Altogether, these data provide a mechanistic explanation for how newborns may cope with an immunosuppressive environment to maintain a certain threshold of innate defenses. However, the same defense mechanisms may be at the expense of the host in conditions of severe infection, suggesting that MIF could represent a potential attractive target for immune-modulating adjunctive therapies for neonatal sepsis.

T-cell activation by treatment of cancer patients with EMD 521873 (Selectikine), an IL-2/anti-DNA fusion protein.

ABSTRACT: BACKGROUND: EMD 521873 (Selectikine or NHS-IL2LT) is a fusion protein consisting of modified human IL-2 which binds specifically to the high-affinity IL-2 receptor, and an antibody specific for both single- and double-stranded DNA, designed to facilitate the enrichment of IL-2 in tumor tissue. METHODS: An extensive analysis of pharmacodynamic (PD) markers associated with target modulation was assessed during a first-in-human phase I dose-escalation trial of Selectikine. RESULTS: Thirty-nine patients with metastatic or locally advanced tumors refractory to standard treatments were treated with increasing doses of Selectikine, and nine further patients received additional cyclophosphamide. PD analysis, assessed during the first two treatment cycles, revealed strong activation of both CD4+ and CD8+ T-cells and only weak NK cell activation. No dose response was observed. As expected, Treg cells responded actively to Selectikine but remained at lower frequency than effector CD4+ T-cells. Interestingly, patient survival correlated positively with both high lymphocyte counts and low levels of activated CD8+ T-cells at baseline, the latter of which was associated with enhanced T-cell responses to the treatment. CONCLUSIONS: The results confirm the selectivity of Selectikine with predominant T-cell and low NK cell activation, supporting follow-up studies assessing the clinical efficacy of Selectikine for cancer patients.

JARID2 is a direct target of the PAX3-FOXO1 fusion protein and inhibits myogenic differentiation of rhabdomyosarcoma cells.

Rhabdomyosarcomas (RMS) are the most frequent soft-tissue sarcoma in children and characteristically show features of developing skeletal muscle. The alveolar subtype is frequently associated with a PAX3-FOXO1 fusion protein that is known to contribute to the undifferentiated myogenic phenotype of RMS cells. Histone methylation of lysine residues controls developmental processes in both normal and malignant cell contexts. Here we show that JARID2, which encodes a protein known to recruit various complexes with histone-methylating activity to their target genes, is significantly overexpressed in RMS with PAX3-FOXO1 compared with the fusion gene-negative RMS (t-test; P < 0.0001). Multivariate analyses showed that higher JARID2 levels are also associated with metastases at diagnosis, independent of fusion gene status and RMS subtype (n = 120; P = 0.039). JARID2 levels were altered by silencing or overexpressing PAX3-FOXO1 in RMS cell lines with and without the fusion gene, respectively. Consistent with this, we demonstrated that JARID2 is a direct transcriptional target of the PAX3-FOXO1 fusion protein. Silencing JARID2 resulted in reduced cell proliferation coupled with myogenic differentiation, including increased expression of Myogenin (MYOG) and Myosin Light Chain (MYL1) in RMS cell lines representative of both the alveolar and embryonal subtypes. Induced myogenic differentiation was associated with a decrease in JARID2 levels and this phenotype could be rescued by overexpressing JARID2. Furthermore, we that showed JARID2 binds to and alters the methylation status of histone H3 lysine 27 in the promoter regions of MYOG and MYL1 and that the interaction of JARID2 at these promoters is dependent on EED, a core component of the polycomb repressive complex 2 (PRC2). Therefore, JARID2 is a downstream effector of PAX3-FOXO1 that maintains an undifferentiated myogenic phenotype that is characteristic of RMS. JARID2 and other components of PRC2 may represent novel therapeutic targets for treating RMS patients.

A protocol guided by transpulmonary thermodilution and lactate levels for resuscitation of patients with severe burns.

Over-resuscitation is deleterious in many critically ill conditions, including major burns. For more than 15 years, several strategies to reduce fluid administration in burns during the initial resuscitation phase have been proposed, but no single or simple parameter has shown superiority. Fluid administration guided by invasive hemodynamic parameters usually resulted in over-resuscitation. As reported in the previous issue of Critical Care, Sánchez-Sánchez and colleagues analyzed the performance of a 'permissive hypovolemia' protocol guided by invasive hemodynamic parameters (PiCCO, Pulsion Medical Systems, Munich, Germany) and vital signs in a prospective cohort over a 3-year period. The authors' results confirm that resuscitation can be achieved with below-normal levels of preload but at the price of a fluid administration greater than predicted by the Parkland formula (2 to 4 mL/kg per% burn). The classic approach based on an adapted Parkland equation may still be the simplest until further studies identify the optimal bundle of resuscitation goals.

Loss of connexin40 is associated with decreased endothelium-dependent relaxations and eNOS levels in the mouse aorta.

Upon agonist stimulation, endothelial cells trigger smooth muscle relaxation through the release of relaxing factors such as nitric oxide (NO). Endothelial cells of mouse aorta are interconnected by gap junctions made of connexin40 (Cx40) and connexin37 (Cx37), allowing the exchange of signaling molecules to coordinate their activity. Wild-type (Cx40(+/+)) and hypertensive Cx40-deficient mice (Cx40(-/-)), which also exhibit a marked decrease of Cx37 in the endothelium, were used to investigate the link between the expression of endothelial connexins (Cx40 and Cx37) and endothelial nitric oxide synthase (eNOS) expression and function in the mouse aorta. With the use of isometric tension measurements in aortic rings precontracted with U-46619, a stable thromboxane A(2) mimetic, we first demonstrate that ACh- and ATP-induced endothelium-dependent relaxations solely depend on NO release in both Cx40(+/+) and Cx40(-/-) mice, but are markedly weaker in Cx40(-/-) mice. Consistently, both basal and ACh- or ATP-induced NO production were decreased in the aorta of Cx40(-/-) mice. Altered relaxations and NO release from aorta of Cx40(-/-) mice were associated with lower expression levels of eNOS in the aortic endothelium of Cx40(-/-) mice. Using immunoprecipitation and in situ ligation assay, we further demonstrate that eNOS, Cx40, and Cx37 tightly interact with each other at intercellular junctions in the aortic endothelium of Cx40(+/+) mice, suggesting that the absence of Cx40 in association with altered Cx37 levels in endothelial cells from Cx40(-/-) mice participate to the decreased levels of eNOS. Altogether, our data suggest that the endothelial connexins may participate in the control of eNOS expression levels and function.