Search

Background: Mediastinal lymph node enlargement is prevalent in patients with idiopathic pulmonary fibrosis (IPF). Studies investigating whether this phenomenon reflects specific immunologic activation are lacking. Methods: Programmed cell death-1 (PD-1)/ programmed cell death ligand-1 (PD-L1) expression in mediastinal lymph nodes and lung tissues was analyzed. PD-1, PD-L1 mRNA expression was measured in tracheobronchial lymph nodes of mice following bleomycin-induced injury on day 14. Finally, the effect of the PD-1 inhibitor, pembrolizumab, in bleomycin-induced pulmonary fibrosis was investigated. Results: We analyzed mediastinal lymph nodes of thirty-three patients (n = 33, IPF: n = 14, lung cancer: n = 10, concomitant IPF and lung cancer: n = 9) and lung tissues of two hundred nineteen patients (n = 219, IPF: 123, controls: 96). PD-1 expression was increased, while PD-L1 expression was decreased, in mediastinal lymph nodes of patients with IPF compared to lung cancer and in IPF lungs compared to control lungs. Tracheobronchial lymph nodes isolated on day 14 from bleomycin-treated mice exhibited increased size and higher PD-1, PD-L1 mRNA levels compared to saline-treated animals. Pembrolizumab blunted bleomycin-induced lung fibrosis, as indicated by reduction in Ashcroft score and improvement in respiratory mechanics. Conclusions: Mediastinal lymph nodes of patients with IPF exhibit differential expression profiles than those of patients with lung cancer indicating distinct immune-mediated pathways regulating fibrogenesis and carcinogenesis. PD-1 expression in mediastinal lymph nodes is in line with lung tissue expression. Lower doses of pembrolizumab might exert antifibrotic effects. Clinical trials aiming to endotype patients based on mediastinal lymph node profiling and accordingly implement targeted therapies such as PD-1 inhibitors are greatly anticipated. [ABSTRACT FROM AUTHOR]

We present a numerical method based on random projections with Gaussian kernels and physics-informed neural networks for the numerical solution of initial value problems (IVPs) of nonlinear stiff ordinary differential equations (ODEs) and index-1 differential algebraic equations (DAEs), which may also arise from spatial discretization of partial differential equations (PDEs). The internal weights are fixed to ones while the unknown weights between the hidden and output layer are computed with Newton's iterations using the Moore–Penrose pseudo-inverse for low to medium scale and sparse QR decomposition with L 2 regularization for medium- to large-scale systems. Building on previous works on random projections, we also prove its approximation accuracy. To deal with stiffness and sharp gradients, we propose an adaptive step-size scheme and address a continuation method for providing good initial guesses for Newton iterations. The "optimal" bounds of the uniform distribution from which the values of the shape parameters of the Gaussian kernels are sampled and the number of basis functions are "parsimoniously" chosen based on bias-variance trade-off decomposition. To assess the performance of the scheme in terms of both numerical approximation accuracy and computational cost, we used eight benchmark problems (three index-1 DAEs problems, and five stiff ODEs problems including the Hindmarsh–Rose neuronal model of chaotic dynamics and the Allen–Cahn phase-field PDE). The efficiency of the scheme was compared against two stiff ODEs/DAEs solvers, namely, ode15s and ode23t solvers of the MATLAB ODE suite as well as against deep learning as implemented in the DeepXDE library for scientific machine learning and physics-informed learning for the solution of the Lotka–Volterra ODEs included in the demos of the library. A software/toolbox in Matlab (that we call RanDiffNet) with demos is also provided. [ABSTRACT FROM AUTHOR]

Background: We have previously shown that SHP2 downregulation may predispose fibroblasts to differentiate into myofibroblasts and proposed a role for SHP2 downregulation in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Recent data have shown that SHP2 localizes to the mitochondrial intercristae, and its overexpression enhances mitochondrial metabolism leading to oxidative stress and senescence. Objective: To determine the effect of SHP2 on fibrotic responses. Methods and Results: Primary mouse lung fibroblasts derived from mice carrying a conditional knock-in mutation (D61G/+), rendering the SHP2 catalytic domain constitutively active, had reduced proliferation (1.6-fold, p < 0.05), migration (2-fold, p < 0.05), as well as reduced responsiveness of TGFB-1 induced fibroblasts-to-myofibroblasts differentiation, compared to wild-type ones. Electron microscope analysis revealed that SHP2 D61G/+ mouse lung fibroblasts were characterized by mitochondrial abnormalities, including swollen mitochondria with disrupted electron-lucent cristae and an increased number of autophagosomes compared to wild-type ones. SHP2 D61G/+ MLFs exhibited increased protein levels of autophagy markers, including LC3B-II and p-62, evidence that was confirmed by immunofluorescence analysis. Mitochondrial function analysis revealed that stable (genotype D61G/+) overexpression of SHP2 led to impaired mitochondrial function, as assessed by decreased mitochondrial membrane potential (1.29-fold, p < 0.05), coupling efficiency (1.82 fold, p < 0.05), oxygen consumption rate (1.9-fold, p < 0.05), and increased reactive oxygen species production both at baseline (1.75-fold, p < 0.05) and following H2O2 stimulation (1.63-fold, p < 0.05) compared to wild-type ones (SHP2+/+). SHP2 D61G/+ mouse lung fibroblasts showed enhanced AMPK activity, as well as decreased activation of the mTORC1 signaling pathway, potentially leading to ineffective mitochondrial metabolism and increased autophagy. Conclusions: SHP2 attenuates fibrotic responses in fibroblast cell lines through negative regulation of mitochondrial metabolism and induction of autophagy. SHP2 activation may represent a promising therapeutic strategy for patients with fibrotic lung diseases. [ABSTRACT FROM AUTHOR]

We localize the sources of brain activity of children with epilepsy based on electroencephalograph (EEG) recordings acquired during a visual discrimination working memory task. For the numerical solution of the inverse problem, with the aid of age‐specific MRI scans processed from a publicly available database, we use and compare three regularization numerical methods, namely the standardized low resolution brain electromagnetic tomography (sLORETA), the weighted minimum norm estimation (wMNE) and the dynamic statistical parametric mapping (dSPM). We show that all three methods provide the same spatio‐temporal patterns of differences between the groups of epileptic and control children. In particular, our analysis reveals statistically significant differences between the two groups in regions of the parietal cortex indicating that these may serve as "biomarkers" for diagnostic purposes and ultimately localized treatment. [ABSTRACT FROM AUTHOR]

Background: High expression levels of Inhibitors of Apoptosis Proteins (IAPs) have been correlated with poor cancer prognosis and block the cell death pathway by interfering with caspase activation. SMAC-mimetics are small-molecule inhibitors of IAPs that mimic the endogenous SMAC and promote the induction of cell death by neutralizing IAPs.Methods: In this study, anti-tumour activity of new SMAC-mimetics Birinapant and AT-406 is evaluated against colorectal adenocarcinoma cells and IAP cross-talk with either oncogenic BRAF or BCL-2, or with the TRAIL are further exploited towards rational combined protocols.Results: It is shown that pre-treatment of SMAC-mimetics followed by their combined treatment with BRAF inhibitors can decrease cell viability, migration and can very efficiently sensitize colorectal tumour cells to apoptosis. Moreover, co-treatment of TRAIL with SMAC-mimetics can efficiently sensitize resistant tumour cells to apoptosis synergistically, as shown by median effect analysis. Finally, Birinapant and AT-406 can synergise with BCL-2 inhibitor ABT-199 to reduce viability of adenocarcinoma cells with high BCL-2 expression.Conclusions: Proposed synergistic rational anticancer combined protocols of IAP antagonists Birinapant and AT-406 in 2D and 3D cultures can be later further exploited in vivo, from precision tumour biology to precision medical oncology. [ABSTRACT FROM AUTHOR]

Abstract: Reactive oxygen species (ROS) were viewed for a long time as unavoidable by-products of normal cell catabolism. This view has recently changed and it is now apparent that ROS generation is a tightly regulated process that plays a central role in cell signaling. Thus, it is known that regulated changes in intracellular ROS levels can induce biochemical signaling processes that control basic cellular functions, such as proliferation and apoptosis which are prevalent in the development of cancer. In this short review, we will try to provide a background to this emerging field by summarizing the biochemistry of ROS-mediated cell signaling and its relation to carcinogenesis. Special emphasis will be focused on the emerging role of the so called “labile” iron (the redox-active form of iron) in ROS-mediated signaling in relation to cancer development. It is tempting to speculate that elucidation of the exact molecular mechanisms that govern ROS-mediated regulation of cell signaling will provide the basis for development of new therapeutic strategies for cancer prevention and treatment. [Copyright &y& Elsevier]

Iron is an essential cofactor for important biological activities and biochemical reactions, including the transport of oxygen via red blood cells and its reduction to water during respiration. While iron's bioavailability is generally limited, pathological accumulation of the metal within tissues aggravates the generation of reactive oxygen species (ROS) and elicits toxic effects, which are mainly related to oxidative stress. Here, we describe the role of iron in ROS-induced toxicity and discuss molecular mechanisms and physiological aspects of ROS- and iron-mediated signaling. In addition, we review our current understanding of the regulation of iron homeostasis at the cellular and systemic levels, and focus on the pathogenesis and management of iron overload disorders. [ABSTRACT FROM AUTHOR]

Metals are necessary for the normal functioning of cells and the survival of organisms. However, exposure to higher than the physiological levels of several metals may lead to tumor development. Although the exact molecular mechanism(s) of metal-induced carcinogenesis is not clear, a vast body of evidence indicates that metal-induced generation of reactive oxygen species (ROS) may play a central role in this process. Two main pathways of ROS-induced effects are discussed in this chapter: (i) increased DNA damage induced either directly or indirectly by impeding DNA repair, and (ii) modulation of nuclear transcriptional factor activities, such as NF-κB and AP-1, through mitogen-activated protein kinases signal transduction mechanisms. [Copyright &y& Elsevier]

Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) is an autosomal dominant inherited disease characterized by arteriovenous malformations and hemorrhage. HHT1 is caused by mutations in ENDOGLIN, which encodes an ancillary receptor for Transforming Growth Factor-β/Bone Morphogenetic Protein-9 expressed in all vascular endothelial cells. Haploinsufficiency is widely accepted as the underlying mechanism for HHT1. However, it remains intriguing that only some, but not all, vascular beds are affected, as these causal gene mutations are present in vasculature throughout the body. Here, we have examined the endoglin expression levels in the blood vessels of multiple organs in mice and in humans. We found a positive correlation between low basal levels of endoglin and the general prevalence of clinical manifestations in selected organs. Endoglin was found to be particularly low in the skin, the earliest site of vascular lesions in HHT1, and even undetectable in the arteries and capillaries of heterozygous endoglin mice. Endoglin levels did not appear to be associated with organ-specific vascular functions. Instead, our data revealed a critical endoglin threshold compatible with the haploinsufficiency model, below which endothelial cells independent of their tissue of origin exhibited abnormal responses to Vascular Endothelial Growth Factor. Our results support the development of drugs promoting endoglin expression as potentially protective. [ABSTRACT FROM AUTHOR]

The value of group work for divorcing families has been documented. In particular, research has shown that support groups for children of divorce may serve an important function in lessening the negative effects of divorce processes on children. At the same time, there are few published protocols as to how to populate and organize these groups. This article describes the model for these groups developed at the Marriage Council of Philadelphia. Details about group organization are given, as is illustrative case material and typical concerns group members work with throughout the life of the group. Reported effects of support group involvement are summarized, and concluding attention is paid to the need to look at ways of intervening in the larger context of the postseparation/divorce process. [ABSTRACT FROM AUTHOR]

Iron is a transition metal and essential constituent of almost all living cells and organisms. As component of various metalloproteins it is involved in critical biochemical processes such as transport of oxygen in tissues, electron transfer reactions during respiration in mitochondria, synthesis and repair of DNA, metabolism of xenobiotics, etc. However, when present in excess within cells and tissues, iron disrupts redox homeostasis and catalyzes the propagation of reactive oxygen species (ROS), leading to oxidative stress. ROS are critical for physiological signaling pathways, but oxidative stress is associated with tissue injury and disease. At the cellular level, oxidative stress may lead to ferroptosis, an iron-dependent form of cell death. In this review, we focus on the intimate relationship between iron metabolism and oxidative stress in health and disease. We discuss aspects of redox- and iron-mediated signaling, toxicity, ferroptotic cell death, homeostatic pathways and pathophysiological implications. • Iron is an essential nutrient but also a potential biohazard. • Iron catalyzes formation of reactive radicals via Fenton chemistry. • Iron overload predisposes to oxidative stress and tissue injury. • Only unshielded labile iron exhibits redox reactivity in vivo. • Labile iron is critical for ferroptosis and can be neutralized by chelating drugs. [ABSTRACT FROM AUTHOR]

Autotaxin (ATX) is a secreted glycoprotein, widely present in biological fluids including blood. ATX catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a growth factor-like, signaling phospholipid. LPA exerts pleiotropic effects mediated by its G-protein-coupled receptors that are widely expressed and exhibit overlapping specificities. Although ATX also possesses matricellular properties, the majority of ATX reported functions in adulthood are thought to be mediated through the extracellular production of LPA. ATX-mediated LPA synthesis is likely localized at the cell surface through the possible interaction of ATX with integrins or other molecules, while LPA levels are further controlled by a group of membrane-associated lipid-phosphate phosphatases. ATX expression was shown to be necessary for embryonic development, and ATX deficient embryos exhibit defective vascular homeostasis and aberrant neuronal system development. In adult life, ATX is highly expressed in the adipose tissue and has been implicated in diet-induced obesity and glucose homeostasis with multiple implications in metabolic disorders. Additionally, LPA has been shown to affect multiple cell types, including stromal and immune cells in various ways. Therefore, LPA participates in many processes that are intricately involved in the pathogenesis of different chronic inflammatory diseases such as vascular homeostasis, skeletal and stromal remodeling, lymphocyte trafficking and immune regulation. Accordingly, increased ATX and LPA levels have been detected, locally and/or systemically, in patients with chronic inflammatory diseases, most notably idiopathic pulmonary fibrosis (IPF), chronic liver diseases, and rheumatoid arthritis. Genetic and pharmacological studies in mice have confirmed a pathogenetic role for ATX expression and LPA signaling in chronic inflammatory diseases, and provided the proof of principle for therapeutic interventions, as exemplified by the ongoing clinical trials for IPF. • Autotaxin is largely responsible for the bulk of extracellular LPA production. • LPA exerts pleiotropic effects mediated by its G-protein-coupled receptors. • Increased ATX and LPA levels have been detected in chronic inflammatory diseases. • Genetic and pharmacologic studies in mice suggest ATX as a therapeutic target. [ABSTRACT FROM AUTHOR]

The underlying pathophysiological mechanisms of hepatic ischemia-reperfusion (I/R) injury have not been completely elucidated. However, it is well known that oxidative stress, caused by a burst of reactive oxygen species (ROS) production during the reperfusion phase, plays a crucial role. A growing body of evidence indicates that the intracellular availability of free iron represents a requirement for ROS-induced adverse effects, as iron catalyzes the generation of highly reactive free radicals. The aim of this study was to examine whether a combination of iron chelators with varying lipophilicity could offer enhanced protection against I/R by diminishing the conversion of weak oxidants, like H 2 O 2 , to extremely reactive ones such as hydroxyl radicals (HO. ). HepG2 cells (hepatocellular carcinoma cell line) were exposed to oxidative stress conditions after pre-treatment with the iron chelators desferrioxamine (DFO) and deferiprone (DFP) alone or in combination. Labile iron pool was estimated using the calcein-acetoxymethyl ester (calcein-AM) method and DNA damage with the comet assay. We subsequently used a rabbit model (male New Zealand white rabbits) of hepatic I/R-induced injury to investigate, by measuring biochemical (ALT, ALT, ALP, γGT) and histological parameters, whether this may be true for in vivo conditions. The combination of a membrane-permeable iron chelator (DFP) with a strong membrane-impermeable one (DFO) raises the level of protection in both hepatic cell lines exposed to oxidative stress conditions and hepatic I/R rabbit model. Our results show that combinations of iron chelators with selected lipophilicity and iron-binding properties may represent a valuable strategy to protect against tissue damage during reperfusion after a period of ischemia. [Display omitted] • Excess production of reactive oxygen species triggers reperfusion injury. • Intracellular free iron catalyzes the generation of highly reactive free radicals. • Chelation of intracellular iron protects hepatic cell lines against oxidative stress. • Iron chelating drugs attenuates liver ischemia/reperfusion injury in rabbits. • Optimum protection is offered by combining membrane-permeable and impermeable drugs. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate in detail the molecular mechanisms by which free fatty acids induce liver toxicity in liver cells. HepG2 and Huh7 human liver cell lines were exposed to varying concentrations of stearate (18:0), oleate (18:1), or mixtures of the two fatty acids, and the effects on cell proliferation, lipid droplet accumulation and induction of endoplasmic reticulum stress and apoptosis were evaluated. It was observed that: (a) stearate, but not oleate, inhibited cell proliferation and induced cell death; (b) stearate-induced cell death had the characteristics of endoplasmic reticulum stress-mediated and mitochondrial-mediated apoptosis; (c) the activation of stearate in the form of stearoyl-CoA was a necessary step for the lipotoxic effect; (d) the capacity of cells to produce and accumulate triacylglycerols in the form of lipid droplets was interrupted following exposure to stearate, whereas it proceeded normally in oleate-treated cells; and (e) the presence of relatively low amounts of oleate protected cells from stearate-induced toxicity and restored the ability of the cells to accumulate triacylglycerols. Our data suggest that interruption of triacylglycerol synthesis in the endoplasmic reticulum, apparently because of the formation of a pool of oversaturated intermediates, represents the key initiating event in the mechanism of saturated fatty acid-induced lipotoxicity. [ABSTRACT FROM AUTHOR]

Background/Aims: Oxidative damage has been reported to be involved in the pathophysiology of chronic kidney disease (CKD) as well as in the pathogenesis of cardiovascular complications of CKD patients. The aim of the present investigation was to evaluate the levels of plasma carbonyl formation, a sensitive marker of enhanced oxidative stress in predialysis, hemodialysis (HD) and peritoneal dialysis (PD) patients. Methods: Plasma samples from 20 apparently healthy control individuals and 127 CKD (stages 2, 3, 4, HD and PD) patients were evaluated by Western blot analysis for the estimation of the levels of protein carbonyl formation. Results: Albumin represented the main plasma carbonylated protein. Increasing carbonylation of albumin was detected along with the severity of CKD, reaching significance at stages 3 and 4 (p < 0.01, compared to healthy controls). The carbonylation of albumin was even higher in the plasma of HD patients (p < 0.001), while in PD patients it was not statistically significant compared to controls (p = 0.224). Conclusions: The data presented in this work indicate that oxidative stress in CKD patients gradually increased during the development of the disease. This stress is probably intensified during HD, but not in PD subjects. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Atrial fibrillation (AF) is the most common arrhythmia encountered in clinical practice representing a major health hazard. Owing to relative inefficacy and side effects of classic antiarrhythmic drugs, current interest has shifted to treatments that target AF substrate. Accumulating evidence suggests that there is a link between oxidative processes and AF. In atrial myocardium during AF, there is substantial oxidative damage that may contribute to atrial remodeling. Several pathophysiological changes that possibly associated with increased oxidative stress in AF have been proposed. These include changes in gene transcriptional profiles and mitochondrial DNA, increased activity of enzymes such as NAD(P)H oxidase and xanthine oxidase, inflammatory processes, activation of the renin–angiotensin system and others. Moreover, oxidative stress is involved in the pathophysiology of several predisposing factors and cardiovascular disorders that correspondingly associated with AF. Preliminary studies using dietary antioxidants such as vitamin C have shown promising results. More evidence has been obtained from studies examining agents with pleiotropic effects, including antioxidant, such as inhibitors of the renin–angiotensin system, statins, corticosteroids and carvedilol. Further investigations are needed in order to elucidate the impact of oxidative stress on atrial remodeling. The clarification of these processes in the setting of AF may lead to the development of novel therapeutic strategies. [Copyright &y& Elsevier]

Abstract: The ability of a number of flavonoids belonging to the flavone, flavonol, flavanone, and flavan-3-ol subclasses to protect cellular DNA from H2O2-induced single-strand breaks and the underlying molecular mechanisms were investigated in this work. Formation of single-strand breaks on nuclear DNA, after exposure of Jurkat cells to continuously generated H2O2 in the presence or absence of the flavonoid compounds, was evaluated by the comet assay (single-cell gel electrophoresis). The results indicate the following structural requirements of flavonoids for effective DNA protection: (a) the ortho-dihydroxy structure in either ring A or ring B, (b) the hydroxyl moiety on position 3 in combination with the oxo group at position 4, and (c) the presence of a C2, C3 double bond in ring C. In contrast to free flavonoids, the ability of complexes of [Fe2+]/[flavonoid] to protect nuclear DNA was decreased as the ratio increased, and the complex was completely inactive when the ratio reached a certain value. Moreover, it was observed that several of the flavonoids tested were able to remove iron from calcein loaded into cells and that this property was in excellent correlation with their ability to protect DNA (Spearman''s correlation coefficient, ρ = 0.9, p = 0.005). The antioxidant (electron donating) capacities of the same flavonoids were also evaluated by a conventional method, but no relation with their DNA-protective ability could be established even when their membrane-penetrating abilities were taken into account (p = 0.64). In conclusion, the results presented in this work strongly support the notion that intracellular binding of iron is responsible for the protection offered by flavonoids against H2O2-induced DNA damage. [Copyright &y& Elsevier]

Investigates the purification and properties of human placental ATP diphosphophydrolase. Purified enzyme as a glycoprotein; Migration of the purified enzyme as a single broad band; Protein sequence analysis; Prevention of the platelet aggregation.

Natural antioxidants, like phenolic acids, possess a unique chemical space that can protect cellular components from oxidative stress. However, their polar carboxylic acid chemotype reduces full intracellular antioxidant potential due to limited diffusion through biological membranes. Here, we have designed and developed a new generation of hydrophobic turn-on fluorescent antioxidant precursors that upon penetration of the cell membrane, reveal a more polar and more potent antioxidant core and simultaneously become fluorescent allowing their intracellular tracking. Their activation is stimulated by polarity alteration by sensing intracellular signals and specifically biothiols. In our design, the carboxylic group of phenolic acids that originally restricts cell entrance is derivatized and conjugated through Copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC) to a coumarin derivative that its fluorescence properties are quenched with a biothiol activatable element. This more hydrophobic precursor readily penetrates cell membrane and once inside the cell the antioxidant core is revealed upon sensing glutathione, its fluorescence is restored in a turn-on manner and the generation of a more polar character traps the molecule inside the cell. This turn-on fluorescent antioxidant precursor that can be applied to phenolic acids, was developed for rosmarinic acid and the conjugate was named as RCG. The selectivity and responsiveness of RCG towards the most abundant biothiols was monitored through a variety of biophysical techniques including UV–Vis, fluorescence and NMR spectroscopy. The electrochemical behavior and free radical scavenging capacity of the precursor RCG and the active compound (RC) was evaluated and compared with the parent compound (rosmarinic acid) through cyclic voltammetry and EPR spectroscopy, respectively. The stability of the newly synthesized bioactive conjugate RC was found significantly higher than the parent rosmarinic acid when exposed to oxygen. Cell uptake experiments were conducted and revealed the internalization of RCG. The degree of intracellular DNA protection offered by RCG and its active drug (RC) on exposure to H 2 O 2 was also evaluated in Jurkat cells. Image 1 • Development of a turn-on fluorescent antioxidant precursor of phenolic acids (RCG). • The electrochemical oxidation profile was revealed with cyclic voltametry. • Free radical scavenging activity was evaluated with EPR spectroscopy. • GSH triggers the release of the active compound. • RCG offers DNA protection against oxidative stress. [ABSTRACT FROM AUTHOR]

Background: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown.Methods: We took advantage of Acvrl1+/- mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1+/- mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans.Results: Acvrl1+/- retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial "tip cells" at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1+/- mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1+/- embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1+/- (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1+/- embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1+/- endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1+/- mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1+/- tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies.Conclusions: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2. [ABSTRACT FROM AUTHOR]

Background Flavonoids possess a rich polypharmacological profile and their biological role is linked to their oxidation state protecting DNA from oxidative stress damage. However, their bioavailability is hampered due to their poor aqueous solubility. This can be surpassed through encapsulation to supramolecular carriers as cyclodextrin (CD). A quercetin- 2HP-β-CD complex has been formerly reported by us. However, once the flavonoid is in its 2HP-β-CD encapsulated state its oxidation potential, its decomplexation mechanism, its potential to protect DNA damage from oxidative stress remained elusive. To unveil this, an array of biophysical techniques was used. Methods The quercetin-2HP-β-CD complex was evaluated through solubility and dissolution experiments, electrochemical and spectroelectrochemical studies (Cyclic Voltammetry), UV–Vis spectroscopy, HPLC-ESI-MS/MS and HPLC-DAD, fluorescence spectroscopy, NMR Spectroscopy, theoretical calculations (density functional theory (DFT)) and biological evaluation of the protection offered against H 2 O 2 -induced DNA damage. Results Encapsulation of quercetin inside the supramolecule's cavity enhanced its solubility and retained its oxidation profile. Although the protective ability of the quercetin-2HP-β-CD complex against H 2 O 2 was diminished, iron serves as a chemical stimulus to dissociate the complex and release quercetin. Conclusions We found that in a quercetin-2HP-β-CD inclusion complex quercetin retains its oxidation profile similarly to its native state, while iron can operate as a chemical stimulus to release quercetin from its host cavity. General significance The oxidation profile of a natural product once it is encapsulated in a supramolecular carrier was unveiled as also it was discovered that decomplexation can be triggered by a chemical stimilus. [ABSTRACT FROM AUTHOR]

Nonsteroidal anti-inflammatory drugs (NSAIDs), including ibuprofen, are amongst the most commonly used medications and produce their anti-inflammatory and analgesic benefits by blocking cyclooxygenase (COX)-2. These drugs also have the potential to prevent and treat cancer and some members of the class including ibuprofen can produce anti-platelet effects. Despite their utility, all NSAIDs are associated with increased risk of cardiovascular side effects which our recent work suggests could be mediated by increased levels of the endogenous NO synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA) leading to reduced endothelial NOS activity and associated endothelial cell dysfunction. ADMA is a cardiotoxic hormone and biomarker of cardiovascular risk whose effects can be prevented by l -arginine. The ibuprofen salt, ibuprofen arginate (Spididol ® ) was created to increase drug solubility but we have previously established that it not only effectively blocks COX-2 but also provides an arginine source able to reverse the effects of ADMA in vitro and in vivo . Here we have gone on to explore whether the formulation of ibuprofen with arginine influences the potency and efficacy of the parent molecule using a range of simple in vitro assays designed to test the effects of NSAIDs on (i) platelet aggregation and (iii) colon cancer cell killing. Our findings demonstrate that ibuprofen arginate retains these key functional effects of NSAIDs with similar or increased potency compared to ibuprofen sodium, further illustrating the potential of ibuprofen arginate as an efficacious drug with the possibility of improved cardiovascular safety. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H 2 O 2 ) acts as a second messenger in signal transduction participating in several redox regulated pathways, including cytokine and growth factor stimulated signals. However, the exact molecular mechanisms underlying these processes remain poorly understood and require further investigation. In this work, using Jurkat T lymphoma cells and primary human umbilical vein endothelial cells, it was observed that changes in intracellular “labile iron” were able to modulate signal transduction in H 2 O 2 –induced apoptosis. Chelation of intracellular labile iron by desferrioxamine rendered cells resistant to H 2 O 2 –induced apoptosis. In order to identify the exact points of iron action, we investigated selected steps in H 2 O 2 -mediated apoptotic pathway, focusing on mitogen activated protein kinases (MAPKs) JNK, p38 and ERK. It was observed that spatiotemporal changes in intracellular labile iron, induced by H 2 O 2 , influenced the oxidation pattern of the upstream MAP3K ASK1 and promoted the sustained activation of JNK-p38 axis in a defined time-dependent context. Moreover, we indicate that H 2 O 2 induced spatiotemporal changes in intracellular labile iron, at least in part, by triggering the destabilization of lysosomal compartments, promoting a concomitant early response in proteins of iron homeostasis. These results raise the possibility that iron-mediated oxidation of distinct proteins may be implicated in redox signaling processes. Since labile iron can be pharmacologically modified in vivo, it may represent a promising target for therapeutic interventions in related pathological conditions. [ABSTRACT FROM AUTHOR]

Motivation: Transient S-sulfenylation of cysteine thiols mediated by reactive oxygen species plays a critical role in pathology, physiology and cell signaling. Therefore, discovery of new S-sulfenylated sites in proteins is of great importance towards understanding how protein function is regulated upon redox conditions. Results: We developed PRESS (PRotEin S-Sulfenylation) web server, a server which can effectively predict the cysteine thiols of a protein that could undergo S-sulfenylation under redox conditions. We envisage that this server will boost and facilitate the discovery of new and currently unknown functions of proteins triggered upon redox conditions, signal regulation and transduction, thus uncovering the role of S-sulfenylation in human health and disease. [ABSTRACT FROM AUTHOR]

A small part of cellular iron, usually called 'labile iron pool' (LIP), is not securely stored and has the potential to catalyse the formation of highly reactive oxygen species. The present work estimated LIP levels in human white cells by using the analytical power of flow cytometry. The method relies essentially on already established principles but has the added value of monitoring LIP in different subpopulations of human blood cells concurrently in a single sample. Examination of 41 apparently healthy individuals revealed a positive correlation between LIP levels and the age of the donors (r=0.656, 0.572 and 0.702 for granulocytes, lymphocytes and monocytes, respectively, p<0.0001), indicating that cells of older individuals are prone to oxidations in conditions of oxidative stress. It is suggested that LIP estimation may represent a valuable tool in examinations searching for links between iron and a variety of oxidative stress-related pathological conditions. [ABSTRACT FROM AUTHOR]

Abstract: Apoptosis represents a physiological form of cell death, the perturbation of which may contribute to the development of several diseases connected with accumulation of unwanted cells or excessive cell loss. We have previously shown that the continuous presence of low concentrations of H2O2 (generated by the action of glucose oxidase) was able to inhibit caspase-mediated apoptosis in Jurkat cells. The main purpose of the present study was to elucidate the exact molecular mechanism(s) underlying this inhibitory action of H2O2. The results presented show that events like outer mitochondrial membrane permeabilization, release of cytochrome c from mitochondria, oligomerization of Apaf-1, and recruitment of procaspase-9 to apoptosomes were taking place normally, but further advancement toward activation of the execution caspases was interrupted when H2O2 was present during the apoptotic process. From the results presented in this work, it emerges that the inhibition of procaspase-9 autoactivation was probably due to the reversible oxidation of sensitive cysteine residues in this molecule. Remarkably, caspase-9 activation and the ensuing caspase cascade proceeded normally in the presence of H2O2 under conditions of iron deprivation, indicating that the inhibition of procaspase-9 activation was an iron-dependent process. Collectively, these results highlighted the potential role of available intracellular iron ions in signaling mechanisms related to apoptotic cell death. [Copyright &y& Elsevier]

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular ‘labile iron pool’ (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at

Abstract: Heat shock protein-70 (Hsp70) is the main heat-inducible member of the 70-kDa family of chaperones that assist cells in maintaining proteins functional under stressful conditions. In the present investigation, the role of Hsp70 in the molecular mechanism of hydrogen peroxide-induced DNA damage to HeLa cells in culture was examined. Stably transfected HeLa cell lines, overexpressing or lacking Hsp70, were created by utilizing constitutive expression of plasmids containing the functional hsp70 gene or hsp70-siRNA, respectively. Compared to control cells, the Hsp70-overexpressing ones were significantly resistant to hydrogen peroxide-induced DNA damage, while Hsp70-depleted cells showed an enhanced sensitivity. In addition, the “intracellular calcein-chelatable iron pool” was determined in the presence or absence of Hsp70 and found to be related to the sensitivity of nuclear DNA to H2O2. It seems likely that the main action of Hsp70, at least in this system, is exerted at the lysosomal level, by protecting the membranes of these organelles against oxidative stress-induced destabilization. Apart from shedding additional light on the mechanistic details behind the action of Hsp70 during oxidative stress, our results indicate that modulation of cellular Hsp70 may represent a way to make cancer cells more sensitive to normal host defense mechanisms or chemotherapeutic drug treatment. [Copyright &y& Elsevier]

In search for compounds, able to protect nuclear DNA in cells exposed to oxidative stress, extracts from olive leaves, olive fruits, olive oil and olive mill waste water were tested by using the “single cell gel electrophoresis” methodology (comet assay). Jurkat cells in culture were exposed to continuously generated hydrogen peroxide (11.8±1.5?µM per min) by direct addition into the growth medium of the appropriate amount of the enzyme “glucose oxidase” in the presence or absence of the tested total extracts. The protective effects of the tested extracts or isolated compounds were evaluated from their ability to decrease hydrogen peroxide-induced formation of single strand breaks in the nuclear DNA, while the toxic effects were estimated from the increase of DNA damage when the extracts or isolated compounds were incubated directly with the cells. Significant protection was observed in extracts from olive oil and olive mill waste water. However, above a concentration of 100?µg/ml olive oil extracts exerted DNA damaging effects by themselves in the absence of any H2O2. Extracts from olive leaves and olive fruits although protective, were also able to induce DNA damage by themselves. Main compounds isolated from the above described total extracts, like oleuropein glucoside, tyrosol, hydroxytyrosol and caffeic acid, were tested in the same experimental system and found to exert cytotoxic (oleuropein glucoside), no effect (tyrosol) or protective effects (hydroxytyrosol and caffeic acid). In conclusion, cytoprotective as well as cytotoxic compounds with potential pharmaceutical properties were detected in extracts from olive oil related sources by using the comet assay methodology. [ABSTRACT FROM AUTHOR]

The patient's position during laparoscopic surgery can have a clinically relevant effect on lower limb and splanchnic circulation; this factor has not yet been investigated with respect to oxidative stress markers. In order to assess this effect, a prospective clinical trial was designed wherein 2 groups of patients were studied. In group A, 15 patients underwent upper abdominal nonhepatobiliary operations (13 modified Nissen fundoplications and 2 Taylor vagotomies) in the head-up position. In group B, 15 patients underwent lower abdominal operations (10 laparoscopic colectomies and 5 inguinal hernia repairs) in the head-down position. The pneumoperitoneum was maintained at 14 mm Hg in all cases. Plasma concentrations of thiobarbituric-acid reactive substances CTBARS), a marker of lipid peroxidation, plasma total antioxidant status (TA5), and serum uric acid concentrations were measured preoperatively, 5 minutes after deflation of the pneumoperitoneum, and 24 hours postoperatively. Aspartate aminotransferase CAST) and alanine aminotransferase (ALT) serum activities were measured preoperatively and 24 hours postoperatively. In group A, there was a significant increase in TBARS levels (p<0.005) immediately after deflation of the pneumoperitoneum and a significant decrease in TAS and uric acid levels Cp<0,005) in the first postoperative day. There was also a significant postoperative elevation in both ALT and AST activities (p<0.001). In group B, no significant increase was found in postoperative TBARS or transaminase levels. TAS and uric acid levels decreased significantly in the first postoperative day Cp<0.05) and Cp<0,005, respectively). In conclusion, these results show that a combination of pneumoperitoneum and the head-up position causes significant increase in lipid peroxidation, decrease in plasma TAS, and increase in transaminases. The mechanism responsible for these events could be the low-flow ischemia-reperfusion syndrome induced by the pneumoperitoneum and aggravated by the head-up position. [ABSTRACT FROM AUTHOR]

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC 50 value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC 50 values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H 2 O 2 but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe 3+ ), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe 3+ ] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form. [ABSTRACT FROM AUTHOR]

The role of endosomal/lysosomal redox-active iron in H2O2-induced nuclear DNA damage as well as in cell proliferation was examined using the iron chelator desferrioxamine (DFO). Transient transfections of HeLa cells with vectors encoding dominant proteins involved in the regulation of various routes of endocytosis (dynamin and Rab5) were used to show that DFO (a potent and rather specific iron chelator) enters cells by fluid-phase endocytosis and exerts its effects by chelating redox-active iron present in the endosomal/lysosomal compartment. Endocytosed DFO effectively protected cells against H2O2-induced DNA damage, indicating the importance of endosomal/lysosomal redox-active iron in these processes. Moreover, exposure of cells to DFO in a range of concentrations (0.1 to 100 μM) inhibited cell proliferation in a fluid-phase endocytosis-dependent manner. Flow cytometric analysis of cells exposed to 100 μM DFO for 24 h showed that the cell cycle was transiently interrupted at the G2/M phase, while treatment for 48 h led to permanent cell arrest. Collectively, the above results clearly indicate that DFO has to be endocytosed by the fluid-phase pathway to protect cells against H2O2-induced DNA damage. Moreover, chelation of iron in the endosomal/lysosomal cell compartment leads to cell cycle interruption, indicating that all cellular labile iron is propagated through this compartment before its anabolic use is possible. [Copyright &y& Elsevier]

Aspects of the molecular mechanism(s) of hydrogen peroxide-induced DNA damage and cell death were studied in the present investigation. Jurkat T-cells in culture were exposed either to low rates of continuously generated H2O2 by the action of glucose oxidase or to a bolus addition of the same agent. In the first case, steady state conditions were prevailing, while in the latter, H2O2 was removed by the cellular defense systems following first order kinetics. By using single-cell gel electrophoresis (also called comet assay), an initial increase in the formation of DNA single-strand breaks was observed in cells exposed to a bolus of 150 μM H2O2. As the H2O2 was exhausted, a gradual decrease in DNA damage was apparent, indicating the existence of an effective repair of single-strand breaks. Addition of 10 ng glucose oxidase in 100 μl growth medium (containing 1.5 × 105 cells) generated 2.0 ± 0.2 μM H2O2 per min. This treatment induced an increase in the level of single-strand breaks reaching the upper limit of detection by the methodology used and continued to be high for the following 6 h. However, when a variety of markers for apoptotic cell death (DNA cell content, DNA laddering, activation of caspases, PARP cleavage) were examined, only bolus additions of H2O2 were able to induce apoptosis, while the continuous presence of this agent inhibited the execution of the apoptotic process no matter whether the inducer was H2O2 itself or an anti-Fas antibody. These observations stress that, apart from the apparent genotoxic and proapoptotic effects of H2O2, it can also exert antiapoptotic actions when present, even at low concentrations, during the execution of apoptosis. [Copyright &y& Elsevier]

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, ‘Demokritos’; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center ‘Alexander Fleming’; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at ‘Andreas Sygros’ Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group''s very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece. [Copyright &y& Elsevier]

Background: The induction of the pneumoperitoneum increases intraabdominal pressure (IAP), causing splanchnic ischemia, whereas its deflation normalizes IAP and splanchnic blood flow. This procedure appears to represent an ischemia-reperfusion model in humans.Methods: Thirty laparoscopic cholecystectomies (LC) were performed in 30 patients with a mean age of 54.6 +/- 15.6 years. A group of 20 patients mean age, 57.3 +/- 9.65 who underwent open cholecystectomy (OC) was also studied. Vein plasma levels of thiobarbituric acid-reactive substances (TBARS), a marker of free radical production; plasma total antioxidant status (TAS); and uric acid (UA) levels were measured preoperatively, 5 min after deflation of the pneumoperitoneum or at the end of operation, and 24 h postoperatively. Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBL) levels were measured preoperatively and 24 h after the operation.Results: In the LC group, significant elevations in the concentration of TBARS were observed in the early postoperative measurements in comparison with the preoperative measurements. TAS and UA levels were decreased significantly 24 h postoperatively compared to preoperative levels. The postoperative levels of AST, ALT, and TBL increased significantly in comparison with the preoperative levels. In the OC group, no alterations in the concentration of TBARS were observed in the postoperative period. The other parameters had changes similar to those recorded for the LC group.Conclusions: Free radical-induced lipid peroxidation associated with a decrease in plasma antioxidant capacity and UA levels as well as altered hepatic function is observed after deflation of the pneumoperitoneum. These results suggest that free radicals are generated at the end of a laparoscopic procedure, possibly as a result of an ischemia-reperfusion phenomenon induced by the inflation and deflation of the pneumoperitoneum. [ABSTRACT FROM AUTHOR]

We present a new method for studying melanin in vivo based on diffuse reflectance spectroscopy of human skin. We find that the optical absorption spectrum of in vivo melanin exhibits an exponential dependence on wavelength, consistent with, but with a higher decay slope than, in vitro results. We offer theoretical justification for this exponential dependence on the basis of a recently proposed model for the structure of eumelanin protomolecules. Moreover, we report on a new method for analysis of diffuse reflectance spectra, which identifies intrinsic differences in absorption spectra between malignant melanoma and dysplastic nevi in vivo. These preliminary results are confirmed both by analysis of our own clinical data as well as by analysis of data from three independent, previously published studies. In particular, we find evidence that the histologic transition from dysplastic nevi to melanoma in situ and then to malignant melanoma is reflected in the melanin absorption spectra. Our results are very promising for the development of techniques for the noninvasive detection of melanoma and, more generally, for the study and characterization of pigmented skin lesions. It is also a promising approach for a better understanding of the biological role, structure, and function of melanin. [ABSTRACT FROM AUTHOR]

Presents a letter to the editor in response to the article "Convergence of atherosclerosis and Alzheimer's disease: inflammation, cholesterol, and misfolded proteins, " by Ivan Casserly and Eric Topol in the April 3, 2004 issue.