Understanding the multifaceted role of inflammatory mediators in ischemic stroke.

The evolution of ischemic brain damage is strongly affected by an inflammatory reaction that involves soluble mediators, such as cytokines and chemokines, and specialized cells activated locally or recruited from the periphery. The immune system affects all phases of the ischemic cascade, from the acute intravascular reaction due to blood flow disruption, to the development of brain tissue damage, repair and regeneration. Increased endothelial expression of adhesion molecules and blood-brain barrier breakdown promotes extravasation and brain recruitment of blood-borne cells, including macrophages, neutrophils, dendritic cells and T lymphocytes, as demonstrated both in animal models and in human stroke. Nevertheless, most anti-inflammatory approaches showing promising results in experimental stroke models failed in the clinical setting. The lack of translation may reside in the redundancy of most inflammatory mediators, exerting both detrimental and beneficial functions. Thus, this review is aimed at providing a better understanding of the dualistic role played by each component of the inflammatory/immune response in relation to the spatio-temporal evolution of ischemic stroke injury.

14.1T magnetic resonance spectroscopic evaluation of metabolic changes in the mouse striatum following transient middle cerebral artery occlusion

Magnetic resonance imaging (MRI) and spectroscopy (MRS) allow establishing theanatomical evolution and neurochemical profiles of ischemic lesions. However onlylimited MRS studies have been reported to-date in mice due to the challenges ofMRS in small organs. The aim of the current work was to study the neurochemicaland imaging sequelae of ischemic stroke in a mouse model in a horizontal bore14.1 Tesla system.ICR-CD1 mice were subjected to 30 minute transient middle cerebral artery occlusion.The extent of the lesion was determined by MRI. The neurochemical profileconsisting of the concentrations of 22 metabolites was measured longitudinallyfollowing the recovery from ischemia at 3, 8 and 24h in the striatum.Our model produced very reproducible striatal lesions which began to appear onT2-weighted images 8h after ischemia. At 24h, they were well established andtheir size correlated with lesions measured by histology. Profound changes couldbe observed in the neurochemical profiles of the core of the striatal lesions as earlyas 3h post-ischemia, in particular, we observed elevated lactate levels, decreases inthe putative neuronal marker N-acetyl-aspartate and in glutamate, and a transienttwo-fold glutamine increase, likely linked to excitotoxic release of glutamate andconversion to glutamine. With further ischemia evolution, other changes appearedat later time-points, mainly decreases of metabolites, consistent with disruption ofcellular function. It is interesting to note that glutamine tended to return to basallevels at 24h.We conclude that early changes in markers of energy metabolism, glutamate excitotoxicityand neuronal viability can be detected with high precision non-invasively inmice following stroke. Such investigations should lead to a better understanding andinsight into the sequential early changes in the brain parenchyma after ischemia,which could be used e.g. for identifying new targets for neuroprotection.

Diurnal and exercise-related variability of haemoglobin and reticulocytes in athletes.

Haemoglobin (Hb) and Reticulocytes (Ret) are measured as indirect markers of doping in athletes. We studied the diurnal variation, the impact of exercise, fluid intake and ambient temperature in athletes on these parameters. Hourly venous blood samples were obtained from 36 male athletes of different disciplines (endurance (END) and non-endurance (NON-END)) over 12 h during a typical training day. Seven inactive subjects served as controls (CON). Hb and Ret were determined. A mixed model procedure was used to analyse the data. At baseline, Hb was similar for all groups, END showed lower Ret than NON-END and CON. Exercise showed a significant impact on Hb (+0.46 g/dl, p<0.001), the effect disappeared approximately 2 h after exercise. Hb decreased over the day by approximately 0.55 g/dl (p<0.01). There was no relevant effect on Ret. Fluid intake and ambient temperature had no significant effect. Hb shows significant diurnal- and exercise related variations. In an anti-doping context, most of these variations are in favour of the athlete. Blood samples taken after exercise might therefore provide reliable results and thus be used for the longitudinal monitoring of athletes if a timeframe for the re-equilibration of vascular volumes is respected.

Redox dysregulation affects the ventral but not dorsal hippocampus: impairment of parvalbumin neurons, gamma oscillations, and related behaviors.

Elevated oxidative stress and alteration in antioxidant systems, including glutathione (GSH) decrease, are observed in schizophrenia. Genetic and functional data indicate that impaired GSH synthesis represents a susceptibility factor for the disorder. Here, we show that a genetically compromised GSH synthesis affects the morphological and functional integrity of hippocampal parvalbumin-immunoreactive (PV-IR) interneurons, known to be affected in schizophrenia. A GSH deficit causes a selective decrease of PV-IR interneurons in CA3 and dendate gyrus (DG) of the ventral but not dorsal hippocampus and a concomitant reduction of beta/gamma oscillations. Impairment of PV-IR interneurons emerges at the end of adolescence/early adulthood as oxidative stress increases or cumulates selectively in CA3 and DG of the ventral hippocampus. Such redox dysregulation alters stress and emotion-related behaviors but leaves spatial abilities intact, indicating functional disruption of the ventral but not dorsal hippocampus. Thus, a GSH deficit affects PV-IR interneuron's integrity and neuronal synchrony in a region- and time-specific manner, leading to behavioral phenotypes related to psychiatric disorders.

Lipoprotein distribution and biological variation of 24S- and 27-hydroxycholesterol in healthy volunteers.

24S- and 27-hydroxycholesterol are obligatory intermediates of cholesterol catabolism and play an important role in the maintenance of whole-body cholesterol homeostasis. Using an HPLC-MS method for oxysterol quantification, the distribution of esterified and unesterified oxysterols in lipoprotein subfractions as well as the influence of daytime, food intake and menstrual cycle on oxysterol concentrations were investigated in healthy volunteers. Moreover, reference intervals for 24S- and 27-hydroxycholesterol in plasma as well as the corresponding levels for 27-hydroxycholesterol in the HDL subfraction were established in 100 healthy volunteers. Both circulating oxysterols are mainly transported in association with HDL and LDL--primarily in the esterified form. No significant diurnal changes and no variations during menstrual cycle of either absolute or cholesterol-related plasma levels were detected. In contrast to 24S-hydroxycholesterol in plasma and 27-hydroxycholesterol in the HDL subfraction, the 95% reference intervals of 27-hydroxycholesterol both in plasma and the non-HDL subfraction were higher in males than in females. The concentrations of 27-hydroxycholesterol in plasma and the non-HDL subfraction showed strong positive correlations with the concentrations of cholesterol, non-HDL cholesterol and triglycerides. Our data on the lipoprotein distribution of oxysterols as well as on their intra- and inter-individual variation set the stage for future clinical studies.

Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Automated Lesion Detection-Based Symptome Mapping In Patients With Sensory Deficits

The human brain is the most complex structure known. With its high number of cells, number of connections and number of pathways it is the source of every thought in the world. It consumes 25% of our oxygen and suffers very fast from a disruption of its supply. An acute event, like a stroke, results in rapid dysfunction referable to the affected area. A few minutes without oxygen and neuronal cells die and subsequently degenerate. Changes in the brains incoming blood flow alternate the anatomy and physiology of the brain. All stroke events leave behind a brain tissue lesion. To rapidly react and improve the prediction of outcome in stroke patients, accurate lesion detection and reliable lesion-based function correlation would be very helpful. With a number of neuroimaging and clinical data of cerebral injured patients this study aims to investigate correlations of structural lesion locations with sensory functions.

Placental growth factor-1 and epithelial haemato-retinal barrier breakdown: potential implication in the pathogenesis of diabetic retinopathy.

AIMS/HYPOTHESIS: Disruption of the retinal pigment epithelial (RPE) barrier contributes to sub-retinal fluid and retinal oedema as observed in diabetic retinopathy. High placental growth factor (PLGF) vitreous levels have been found in diabetic patients. This work aimed to elucidate the influence of PLGF-1 on a human RPE cell line (ARPE-19) barrier in vitro and on normal rat eyes in vivo. METHODS: ARPE-19 permeability was measured using transepithelial resistance and inulin flux under stimulation of PLGF-1, vascular endothelial growth factor (VEGF)-E and VEGF 165. Using RT-PCR, we evaluated the effect of hypoxic conditions or insulin on transepithelial resistance and on PLGF-1 and VEGF receptors. The involvement of mitogen-activated protein kinase (MEK, also known as MAPK)/extracellular signal-regulated kinase (ERK, also known as EPHB2) signalling pathways under PLGF-1 stimulation was evaluated by western blot analysis and specific inhibitors. The effect of PLGF-1 on the external haemato-retinal barrier was evaluated after intravitreous injection of PLGF-1 in the rat eye; evaluation was by semi-thin analysis and zonula occludens-1 immunolocalisation on flat-mounted RPE. RESULTS: In vitro, PLGF-1 induced a reversible decrease of transepithelial resistance and enhanced tritiated inulin flux. These effects were specifically abolished by an antisense oligonucleotide directed at VEGF receptor 1. Exposure of ARPE-19 cells to hypoxic conditions or to insulin induced an upregulation of PLGF-1 expression along with increased transcellular permeability. The PLGF-1-induced RPE cell permeability involved the MEK signalling pathway. Injection of PLGF-1 in the rat eye vitreous induced an opening of the RPE tight junctions with subsequent sub-retinal fluid accumulation, retinal oedema and cytoplasm translocation of junction proteins. CONCLUSIONS/INTERPRETATION: Our results indicate that PLGF-1 may be a potential regulation target for the control of diabetic retinal and macular oedema.

Gelatinase expression and proteolytic activity in giant-cell arteritis

Objectives: Gelatinases (MMP2 and MMP9) are expressed in giant-cell arteritis (GCA) and are thought to play a role in vessel disruption. However, their activation status and enzymatic activity have not been evaluated. Our aim was to investigate the distribution and proteolytic activity of gelatinases in GCA lesions at different stages. Methods: Expression of MMP2, MMP9, MMP2-activator MMP14 and their natural inhibitors TIMP1 and TIMP2 was determined by real-time PCR and immunohistochemistry in temporal artery sections from 46 patients and 12 controls. MMP activation status and enzymatic activity were assessed by gelatin and film in situ zymography. Results: Vascular smooth muscle cells from normal specimens constitutively expressed pro-MMP2 and its inhibitor TIMP2 with no resulting proteolytic activity. In GCA MMP2, MMP9 and MMP14 were strongly expressed in their active form by infiltrating leucocytes. Inflamed arteries also expressed TIMP1 and TIMP2. However, the MMP9/TIMP1 and MMP2/TIMP2 ratios were higher in patients compared with controls, indicating an increased proteolytic balance in GCA which was confirmed by in situ zymography. Maximal gelatinase expression and activity occurred at the granulomatous areas surrounding the internal elastic lamina (IEL). Myointimal cells also expressed MMPs and exhibited proteolytic activity, suggesting a role for gelatinases in vascular remodelling and repair. Conclusions: GCA lesions show intense expression of gelatinases. Activators and inhibitors are regulated to yield enhanced gelatinase activation and proteolytic activity. Distribution of expression and proteolytic activity suggests that gelatinases have a major role not only in the progression of inflammatory infiltrates and vessel destruction but also in vessel repair.

Polyunsaturated aldehydes from large phytoplankton of the Atlantic Ocean Surface (42ºN to 33ºS)

Polyunsaturated aldehydes (PUAs) are organic compounds mainly produced by diatoms, after cell wounding. These compounds are increasingly reported as teratogenic for species of grazers and deleterious for phytoplanktonic species, but there is still scarce information regarding concentration ranges and the composition of PUAs in the open ocean. In this study, we analyzed the spatial distribution and the type of aldehydes produced by the large-sized (>10 μm) phytoplankton in the Atlantic Ocean surface. Analyses were conducted on PUAs released after mechanical disruption of the phytoplankton cells, referred to here as potential PUAs (pPUAs). Results show the ubiquitous presence of pPUA in the open ocean, including upwelling areas, as well as oligotrophic gyres. Total pPUA concentrations ranged from zero to 4.18 pmol from cells in 1 L. Identified PUAs were heptadienal, octadienal and decadienal, with heptadienal being the most common (79% of total stations). PUA amount and composition across the Atlantic Ocean was mainly related to the nitrogen:phosphorus ratio, suggesting nutrient-driven mechanisms of PUA production. Extending the range of trophic conditions considered by adding data reported for productive coastal waters, we found a pattern of PUA variation in relation to trophic status.

Cancer chronotherapeutics: experimental, theoretical, and clinical aspects.

The circadian timing system controls cell cycle, apoptosis, drug bioactivation, and transport and detoxification mechanisms in healthy tissues. As a consequence, the tolerability of cancer chemotherapy varies up to several folds as a function of circadian timing of drug administration in experimental models. Best antitumor efficacy of single-agent or combination chemotherapy usually corresponds to the delivery of anticancer drugs near their respective times of best tolerability. Mathematical models reveal that such coincidence between chronotolerance and chronoefficacy is best explained by differences in the circadian and cell cycle dynamics of host and cancer cells, especially with regard circadian entrainment and cell cycle variability. In the clinic, a large improvement in tolerability was shown in international randomized trials where cancer patients received the same sinusoidal chronotherapy schedule over 24h as compared to constant-rate infusion or wrongly timed chronotherapy. However, sex, genetic background, and lifestyle were found to influence optimal chronotherapy scheduling. These findings support systems biology approaches to cancer chronotherapeutics. They involve the systematic experimental mapping and modeling of chronopharmacology pathways in synchronized cell cultures and their adjustment to mouse models of both sexes and distinct genetic background, as recently shown for irinotecan. Model-based personalized circadian drug delivery aims at jointly improving tolerability and efficacy of anticancer drugs based on the circadian timing system of individual patients, using dedicated circadian biomarker and drug delivery technologies.

Stab wounds in a Swiss emergency department: a series of 80 consecutive cases.

Background: Interpersonal violence is a worldwide social reality which seems to increasingly affect even the safest of countries, such as Switzerland. In this country, road traffic accidents, as well as professional and recreational activities, are the main providers of trauma-related injuries. The incidence of penetrative trauma related to stab wounds seems to be regularly increasing in our ED. The question arises of whether our strategies in trauma management are adapted to deal efficiently with these injuries.Methods: To answer this question, the study analysed patients admitted for intentional penetrative injuries in a tertiary urban emergency department (ED) during a 23 month period. Demographics, conditions of the assault, injury type and treatments applied were analysed.Results: Eighty patients admitted due to an intentional penetrating trauma accounted for 0.2% of the surgical practice of our ED. The assault occurred equally in a public or a private context, mainly affecting young males during the night and the weekend. Sixty six patients (83%) were treated as out-patients. Only 10 patients needed surgery. None of them required damage control surgery. No patient died and the mean hospital stay was 5.5 days.Conclusions: The prevalence of stab wounds in Switzerland is low. These injuries rarely need complex, surgical procedures. Observational strategies should be considered according to the patient status.

VP22 light controlled delivery of oligonucleotides to ocular cells in vitro and in vivo.

PURPOSE: To study VP22 light controlled delivery of antisense oligonucleotide (ODN) to ocular cells in vitro and in vivo. METHODS: The C-terminal half of VP22 was expressed in Escherichia coli, purified and mixed with 20 mer phosphorothioate oligonucleotides (ODNs) to form light sensitive complex particles (vectosomes). Uptake of vectosomes and light induced redistribution of ODNs in human choroid melanoma cells (OCM-1) and in human retinal pigment epithelial cells (ARPE-19) were studied by confocal and electron microscopy. The effect of vectosomes formed with an antisense ODN corresponding to the 3'-untranslated region of the human c-raf kinase gene on the viability and the proliferation of OCM-1 cells was assessed before and after illumination. Cells incubated with vectosomes formed with a mismatched ODN, a free antisense ODN or a free mismatched ODN served as controls. White light transscleral illumination was carried out 24 h after the intravitreal injection of vectosomes in rat eyes. The distribution of fluorescent vectosomes and free fluorescent ODN was evaluated on cryosections by fluorescence microscopy before, and 1 h after illumination. RESULTS: Overnight incubation of human OCM-1 and ARPE-19 cells with vectosomes lead to intracellular internalization of the vectosomes. When not illuminated, internalized vectosomes remained stable within the cell cytoplasm. Disruption of vectosomes and release of the complexed ODN was induced by illumination of the cultures with a cold white light or a laser beam. In vitro, up to 60% inhibition of OCM-1 cell proliferation was observed in illuminated cultures incubated with vectosomes formed with antisense c-raf ODN. No inhibitory effect on the OCM-1 cell proliferation was observed in the absence of illumination or when the cells are incubated with a free antisense c-raf ODN and illuminated. In vivo, 24 h after intravitreal injection, vectosomes were observed within the various retinal layers accumulating in the cytoplasm of RPE cells. Transscleral illumination of the injected eyes with a cold white light induced disruption of the vectosomes and a preferential localization of the "released" ODNs within the cell nuclei of the ganglion cell layer, the inner nuclear layer and the RPE cells. CONCLUSIONS: In vitro, VP22 light controlled delivery of ODNs to ocular cells nuclei was feasible using white light or laser illumination. In vivo, a single intravitreal injection of vectosomes, followed by transscleral illumination allowed for the delivery of free ODNs to retinal and RPE cells.

Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.

Interactions of cell-autonomous circadian oscillators with diurnal cycles govern the temporal compartmentalization of cell physiology in mammals. To understand the transcriptional and epigenetic basis of diurnal rhythms in mouse liver genome-wide, we generated temporal DNA occupancy profiles by RNA polymerase II (Pol II) as well as profiles of the histone modifications H3K4me3 and H3K36me3. We used these data to quantify the relationships of phases and amplitudes between different marks. We found that rhythmic Pol II recruitment at promoters rather than rhythmic transition from paused to productive elongation underlies diurnal gene transcription, a conclusion further supported by modeling. Moreover, Pol II occupancy preceded mRNA accumulation by 3 hours, consistent with mRNA half-lives. Both methylation marks showed that the epigenetic landscape is highly dynamic and globally remodeled during the 24-hour cycle. While promoters of transcribed genes had tri-methylated H3K4 even at their trough activity times, tri-methylation levels reached their peak, on average, 1 hour after Pol II. Meanwhile, rhythms in tri-methylation of H3K36 lagged transcription by 3 hours. Finally, modeling profiles of Pol II occupancy and mRNA accumulation identified three classes of genes: one showing rhythmicity both in transcriptional and mRNA accumulation, a second class with rhythmic transcription but flat mRNA levels, and a third with constant transcription but rhythmic mRNAs. The latter class emphasizes widespread temporally gated posttranscriptional regulation in the mouse liver.