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".

A critical role of autophagy in antileukemia/lymphoma effects of APO866, an inhibitor of NAD biosynthesis.

APO866, an inhibitor of NAD biosynthesis, exhibits potent antitumor properties in various malignancies. Recently, it has been shown that APO866 induces apoptosis and autophagy in human hematological cancer cells, but the role of autophagy in APO866-induced cell death remains unclear. Here, we report studies on the molecular mechanisms underlying APO866-induced cell death with emphasis on autophagy. Treatment of leukemia and lymphoma cells with APO866 induced both autophagy, as evidenced by an increase in autophagosome formation and in SQSTM1/p62 degradation, but also increased caspase activation as revealed by CASP3/caspase 3 cleavage. As an underlying mechanism, APO866-mediated autophagy was found to deplete CAT/catalase, a reactive oxygen species (ROS) scavenger, thus promoting ROS production and cell death. Inhibition of autophagy by ATG5 or ATG7 silencing prevented CAT degradation, ROS production, caspase activation, and APO866-induced cell death. Finally, supplementation with exogenous CAT also abolished APO866 cytotoxic activity. Altogether, our results indicated that autophagy is essential for APO866 cytotoxic activity on cells from hematological malignancies and also indicate an autophagy-dependent CAT degradation, a novel mechanism for APO866-mediated cell killing. Autophagy-modulating approaches could be a new way to enhance the antitumor activity of APO866 and related agents.

Targeting vascular NADPH oxidase 1 blocks tumor angiogenesis through a PPARα mediated mechanism.

Reactive oxygen species, ROS, are regulators of endothelial cell migration, proliferation and survival, events critically involved in angiogenesis. Different isoforms of ROS-generating NOX enzymes are expressed in the vasculature and provide distinct signaling cues through differential localization and activation. We show that mice deficient in NOX1, but not NOX2 or NOX4, have impaired angiogenesis. NOX1 expression and activity is increased in primary mouse and human endothelial cells upon angiogenic stimulation. NOX1 silencing decreases endothelial cell migration and tube-like structure formation, through the inhibition of PPARα, a regulator of NF-κB. Administration of a novel NOX-specific inhibitor reduced angiogenesis and tumor growth in vivo in a PPARα dependent manner. In conclusion, vascular NOX1 is a critical mediator of angiogenesis and an attractive target for anti-angiogenic therapies.

Oxidative stress in aging: advances in proteomic approaches

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual"s Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.

The oxidative potential of differently charged silver and gold nanoparticles on three human lung epithelial cell types

Background: Nanoparticle (NPs) functionalization has been shown to affect their cellular toxicity. To study this, differently functionalized silver (Ag) and gold (Au) NPs were synthesised, characterised and tested using lung epithelial cell systems. Mehtods: Monodispersed Ag and Au NPs with a size range of 7 to 10 nm were coated with either sodium citrate or chitosan resulting in surface charges from ¿50 mV to +70 mV. NP-induced cytotoxicity and oxidative stress were determined using A549 cells, BEAS-2B cells and primary lung epithelial cells (NHBE cells). TEER measurements and immunofluorescence staining of tight junctions were performed to test the growth characteristics of the cells. Cytotoxicity was measured by means of the CellTiter-Blue ® and the lactate dehydrogenase assay and cellular and cell-free reactive oxygen species (ROS) production was measured using the DCFH-DA assay. Results: Different growth characteristics were shown in the three cell types used. A549 cells grew into a confluent mono-layer, BEAS-2B cells grew into a multilayer and NHBE cells did not form a confluent layer. A549 cells were least susceptible towards NPs, irrespective of the NP functionalization. Cytotoxicity in BEAS-2B cells increased when exposed to high positive charged (+65-75 mV) Au NPs. The greatest cytotoxicity was observed in NHBE cells, where both Ag and Au NPs with a charge above +40 mV induced cytotoxicity. ROS production was most prominent in A549 cells where Au NPs (+65-75 mV) induced the highest amount of ROS. In addition, cell-free ROS measurements showed a significant increase in ROS production with an increase in chitosan coating. Conclusions: Chitosan functionalization of NPs, with resultant high surface charges plays an important role in NP-toxicity. Au NPs, which have been shown to be inert and often non-cytotoxic, can become toxic upon coating with certain charged molecules. Notably, these effects are dependent on the core material of the particle, the cell type used for testing and the growth characteristics of these cell culture model systems.

Application of RNAi to silence tartrate-resistant acid phosphatase: unexpected effects on the monocyte-macrophage lineage

Tartraatti-resistentin happaman fosfataasin hiljentäminen RNAi menetelmällä: odottamaton vaikutus monosyytti-makrofagi linjan soluissa RNA interferenssi (RNAi) eli RNA:n hiljentyminen löydettiin ensimmäisenä kasveissa, ja 2000-luvulla RNAi menetelmä on otettu käyttöön myös nisäkässoluissa. RNAi on mekanismi, jossa lyhyet kaksi juosteiset RNA molekyylit eli siRNA:t sitoutuvat proteiinikompleksiin ja sitoutuvat komplementaarisesti proteiinia koodaavaan lähetti RNA:han katalysoiden lähetti RNA:n hajoamisen. Tällöin RNA:n koodaamaa proteiinia ei solussa tuoteta. Tässä työssä on RNA interferenssi menetelmän avuksi kehitetty uusi siRNA molekyylien suunnittelualgoritmi siRNA_profile, joka etsii lähetti RNA:sta geenin hiljentämiseen sopivia kohdealueita. Optimaalisesti suunnitellulla siRNA molekyylillä voi olla mahdollista saavuttaa pitkäaikainen geenin hiljeneminen ja spesifinen kohdeproteiinin määrän aleneminen solussa. Erilaiset kemialliset modifikaatiot, mm. 2´-Fluoro-modifikaatio, siRNA molekyylin riboosirenkaassa lisäsivät siRNA molekyylin stabiilisuutta veren plasmassa sekä siRNA molekyylin tehokkuutta. Nämä ovat tärkeitä siRNA molekyylien ominaisuuksia kun RNAi menetelmää sovelletaan lääketieteellisiin tarkoituksiin. Tartraatti-resistentti hapan fosfataasi (TRACP) on entsyymi, joka esiintyy luunsyöjäsoluissa eli osteoklasteissa, antigeenejä esittelevissä dendiriittisissä soluissa sekä eri kudosten makrofageissa, jotka ovat syöjäsoluja. TRACP entsyymin biologista tehtävää ei ole saatu selville, mutta oletetaan että TRACP entsyymin kyvyllä tuottaa reaktiivisia happiradikaaleja on tehtävä sekä luuta hajoittavissa osteoklasteissa sekä antigeenia esittelevissä dendriittisissä soluissa. Makrofageilla, jotka yliekpressoivat TRACP entsyymiä, on myös solunsisäinen reaktiivisten happiradikaalien tuotanto sekä bakteerin tappokyky lisääntynyt. TRACP-geenin hiljentämiseen tarkoitetut spesifiset DNA ja siRNA molekyylit aiheuttivat monosyytti-makrofagilinjan soluviljelymallissa TRACP entsyymin tuoton lisääntymistä odotusten vastaisesti. DNA ja RNA molekyylien vaikutusta TRACP entsyymin tuoton lisääntymiseen tutkittiin myös Tolllike reseptori 9 (TLR9) poistogeenisestä hiirestä eristetyissä monosyyttimakrofaagisoluissa. TRACP entsyymin tuoton lisääntyminen todettiin sekvenssistä ja TLR9:stä riippumattomaksi vasteeksi solun ulkopuolisia DNA ja RNA molekyylejä vastaan. Havainto TRACP entsyymin tuoton lisääntymisestä viittaa siihen, että TRACP entsyymillä on tehtävä solun immuunipuolustusjärjestelmässä.

Probing functional groups at the gas-aerosol interface using heterogeneous titration reactions: a tool for predicting aerosol health effects?

The complex chemical and physical nature of combustion and secondary organic aerosols (SOAs) in general precludes the complete characterization of both bulk and interfacial components. The bulk composition reveals the history of the growth process and therefore the source region, whereas the interface controls--to a large extent--the interaction with gases, biological membranes, and solid supports. We summarize the development of a soft interrogation technique, using heterogeneous chemistry, for the interfacial functional groups of selected probe gases [N(CH(3))(3), NH(2)OH, CF(3)COOH, HCl, O(3), NO(2)] of different reactivity. The technique reveals the identity and density of surface functional groups. Examples include acidic and basic sites, olefinic and polycyclic aromatic hydrocarbon (PAH) sites, and partially and completely oxidized surface sites. We report on the surface composition and oxidation states of laboratory-generated aerosols and of aerosols sampled in several bus depots. In the latter case, the biomarker 8-hydroxy-2'-deoxyguanosine, signaling oxidative stress caused by aerosol exposure, was isolated. The increase in biomarker levels over a working day is correlated with the surface density N(i)(O3) of olefinic and/or PAH sites obtained from O(3) uptakes as well as with the initial uptake coefficient, γ(0), of five probe gases used in the field. This correlation with γ(0) suggests the idea of competing pathways occurring at the interface of the aerosol particles between the generation of reactive oxygen species (ROS) responsible for oxidative stress and cellular antioxidants.

Mitochondrial DNA mutations induce mitochondrial dysfunction, apoptosis and sarcopenia in skeletal muscle of mitochondrial DNA mutator mice

Background: Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established. Methodology/Principal Findings: We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase c, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Dym). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. Conclusions/Significance: These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

The biological basis of the aging process

El procés biològic bàsic subjacent de l’envelliment va ésser avançat per la teoria de l’envelliment basada en els radicals lliures l’any 1954: la reacció dels radicals lliures actius, produïts fisiològicament en l’organisme, amb els constituents cel·lulars inicia els canvis associats a l’envelliment. La implicació dels radicals lliures en l’envelliment està relacionada amb el seu paper clau en l’origen i l’evolució de la vida. La informació disponible avui en dia ens mostra que la composició específica de les macromolècules cel·lulars (proteïnes, àcids nucleics, lípids i carbohidrats) en les espècies animals longeves tenen intrínsicament una resistència elevada a la modificació oxidativa, la qual cosa probablement contribueix a la longevitat superior d’aquestes espècies. Les espècies longeves també mostren unes taxes reduïdes de producció de radicals lliures i de lesió oxidativa. D’altra banda, la restricció dietària disminueix la producció de radicals lliures i la lesió molecular oxidativa. Aquests canvis estan directament associats a la reducció de la ingesta de proteïnes dels animals sotmesos a restricció, que alhora sembla que són deguts específicament a la reducció de la ingesta de metionina. En aquesta revisió s’emfatitza que una taxa baixa de generació de lesió endògena i una resistència intrínsecament elevada a la modificació de les macromolècules cel·lulars són trets clau de la longevitat de les espècies animals.

Xanthine oxidoreductase regulates macrophage IL1β secretion upon NLRP3 inflammasome activation.

Activation of the NLRP3 inflammasome by microbial ligands or tissue damage requires intracellular generation of reactive oxygen species (ROS). We present evidence that macrophage secretion of IL1β upon stimulation with ATP, crystals or LPS is mediated by a rapid increase in the activity of xanthine oxidase (XO), the oxidized form of xanthine dehydrogenase, resulting in the formation of uric acid as well as ROS. We show that XO-derived ROS, but not uric acid, is the trigger for IL1β release and that XO blockade results in impaired IL1β and caspase1 secretion. XO is localized to both cytoplasmic and mitochondrial compartments and acts upstream to the PI3K-AKT signalling pathway that results in mitochondrial ROS generation. This pathway represents a mechanism for regulating NLRP3 inflammasome activation that may have therapeutic implications in inflammatory diseases.

Mitochondrial DNA damage and animal longevity: insights from comparative studies

Chemical reactions in living cells are under strict enzyme control and conform to a tightly regulated metabolic program. However, uncontrolled and potentially deleterious endogenous reactions occur, even under physiological conditions. Aging, in this chemical context, could be viewed as an entropic process, the result of chemical side reactions that chronically and cumulatively degrade the function of biological systems. Mitochondria are a main source of reactive oxygen species (ROS) and chemical sidereactions in healthy aerobic tissues and are the only known extranuclear cellular organelles in animal cells that contain their own DNA (mtDNA). ROS can modify mtDNA directly at the sugar-phosphate backbone or at the bases, producing many different oxidatively modified purines and pyrimidines, as well as single and double strand breaks and DNA mutations. In this scenario, natural selection tends to decrease the mitochondrial ROS generation, the oxidative damage to mtDNA, and the mitochondrial mutation rate in long-lived species, in agreement with the mitochondrial oxidative stress theory of aging.

Physico-chemical characterization and oxidative reactivity evaluation of aged brake wear particles

Brake wear dust is a significant component of traffic emissions and has been linked to adverse health effects. Previous research found a strong oxidative stress response in cells exposed to freshly generated brake wear dust. We characterized aged dust collected from passenger vehicles, using microscopy and elemental analyses. Reactive oxygen species (ROS) generation was measured with acellular and cellular assays using 2′7-dichlorodihydrofluorescein dye. Microscopy analyses revealed samples to be heterogeneous particle mixtures with few nanoparticles detected. Several metals, primarily iron and copper, were identified. High oxygen concentrations suggested that the elements were oxidized. ROS were detected in the cell-free fluorescent test, while exposed cells were not dramatically activated by the concentrations used. The fact that aged brake wear samples have lower oxidative stress potential than fresh ones may relate to the highly oxidized or aged state of these particles, as well as their larger size and smaller reactive surface area.

GD3 Synthase Overexpression Sensitizes Hepatocarcinoma Cells to Hypoxia and Reduces Tumor Growth by Suppressing the cSrc/NF-κB Survival Pathway

Abstract Background: Hypoxia-mediated HIF-1a stabilization and NF-kB activation play a key role in carcinogenesis by fostering cancer cell survival, angiogenesis and tumor invasion. Gangliosides are integral components of biological membranes with an increasingly recognized role as signaling intermediates. In particular, ganglioside GD3 has been characterized as a proapoptotic lipid effector by promoting cell death signaling and suppression of survival pathways. Thus, our aim was to analyze the role of GD3 in hypoxia susceptibility of hepatocarcinoma cells and in vivo tumor growth. Methodology/Principal Findings: We generated and characterized a human hepatocarcinoma cell line stably expressing GD3 synthase (Hep3B-GD3), which catalyzes the synthesis of GD3 from GM3. Despite increased GD3 levels (2-3 fold), no significant changes in cell morphology or growth were observed in Hep3B-GD3 cells compared to wild type Hep3B cells under normoxia. However, exposure of Hep3B-GD3 cells to hypoxia (2% O2) enhanced reactive oxygen species (ROS) generation, resulting in decreased cell survival, with similar findings observed in Hep3B cells exposed to increasing doses of exogenous GD3. In addition, hypoxia-induced c-Src phosphorylation at tyrosine residues, NF-kB activation and subsequent expression of Mn-SOD were observed in Hep3B cells but not in Hep3B-GD3 cells. Moreover, MnTBAP, an antioxidant with predominant SOD mimetic activity, reduced ROS generation, protecting Hep3B-GD3 cells from hypoxia-induced death. Finally, lower tumor growth, higher cell death and reduced Mn-SOD expression were observed in Hep3B-GD3 compared to Hep3B tumor xenografts. Conclusion: These findings underscore a role for GD3 in hypoxia susceptibility by disabling the c-Src/NF-kB survival pathway resulting in lower Mn-SOD expression, which may be of relevance in hepatocellular carcinoma therapy.

Role of basic calcium phosphate crystals in osteoarthritis

L'arthrose est une maladie dégénérative des articulations due à une dégradation progressive du cartilage. La calcification de l'articulation (essentiellement due à des dépôts de cristaux de phosphate de calcium basique -cristaux BCP-) est une caractéristique de cette maladie. Cependant, le rôle des cristaux BCP reste à déterminer. Nous avons tout d'abord déterminé en utilisant des cultures primaires de chondrocytes que les cristaux de BCP induisaient la production de la cytokine IL-6, via une signalisation intracellulaire implicant les kinase Syk, PI3 et Jak et Stat3. Les cristaux de BCP induisent également la perte de protéoglycanes et l'expression de IL-6 dans des explants de cartlage humain et ces deux effets peuvent être bloqués par un inhibiteur de IL-6, le Tocilizumab. Par ailleurs, nous avons trouvé que l'IL-6 ajouté à des chondrocytes, favorisait la formation de cristax de BCP et augmentait l'expression de gènes impliqués dans le processus de minéralisation : Ank (codant pour un transporteur de pyrophooshate), Annexin5 (codant pour un canal calcique) et Pit-1 (codant pour un transporteur de phoshate). In vivo, les cristaux de BCP injectés dans l'articulation de souris induisent une érosion du cartilage. Dans un modèle murin d'arthrose du genou induit par ménisectomie, nous avons observé la formation progressive de cristaux de BCP. Fait intéressant, la présence de ces cristaux dans l'articulation précédait la destruction du cartilage. Un agent susceptible de bloquer les calcifications tel que le sodium thiosulfate (STS), administré à des souris ménisectomisées, inhibait le dépôt intra-articulaire de ces cristaux ainsi que l'érosion du cartilage. Nous avons identifié ainsi un cercle vicieux dans l'arthrose, les cristaux induisant l'interleukine-6 et l'interleukine-6 induisant la formation de ces cristaux. Nous avons étudié si on pouvait bloquer cette boucle cristaux de BCP-IL6 soit par des agents décalcifiants, soit par des inhibiteurs d'IL-6. In vitro, des anticorps anti IL- 6 ou des inhibiteurs de signalisation, inhibaient significativement IL-6 et la minéralisation induite par IL-6. De même le STS inhibait la formation de ces cristaux et la production de l'IL-6. Tout récemment, nous avons trouvé que des inhibiteurs de la xanthine oxidoréductase étaient aussi capables d'inhiber à la fois la production d'IL-6 et la minéralization des chondrocytes. Finalement, nous avons pu exclure un rôle du système IL-1 dans le modèle d'arthrose induite par ménisectomie, les souris déficientes pour IL-1a/ß, MyD88 et l'inflammasome NLRP3 n'étant pas protégées dans ce modèle d'arthrose. L'ensemble de nos résultats montre que les cristaux BCP sont pathogéniques dans l'arthrose et qu'un inhibiteur de minéralisation tel que le STS ou un inhibiteur de l'interleukine-6 constitueraient des nouvelles thérapies pour l'arthrose. -- Osteoarthritis (OA), the most common degenerative disorder of the joints, results from an imbalance between the breakdown and repair of the cartilage and surrounding articular structures. Joint calcification (essentially due to basic calcium phosphate (BCP) crystal deposition) is a characteristic feature of OA. However, the role of BCP crystal deposition in the pathogenesis of OA remains unclear[1][1]. We first demonstrated that in primary murine chondrocytes exogenous BCP crystals led to IL-6 up-modulation and that BCP crystal signaling pathways involved Syk and PI3 kinases, and also gp130 associated molecules, Jak2 and Stat3. BCP crystals also induced proteoglycan loss and IL-6 expression in human cartilage expiants, (which were significantly reduced by an IL-6 inhibitor). In addition, we found that in chondrocytes exogenous IL-6 promoted calcium-containing crystal formation and up- regulation of genes codifying for proteins involved in the calcification process: the inorganic pyrophosphate transport channel Ank, the calcium channel Annexinö and the sodium/phosphate cotransporter Piti. In vivo, BCP crystals injected into murine knee joints induced cartilage erosion. In the menisectomy model, increasing deposits, identified as BCP crystals, were progressively observed around the joint before cartilage erosion. These deposits strongly correlated with cartilage degradation and IL-6 expression. These results demonstrated that BCP crystals deposition and IL-6 production are mutually reinforcing in the osteoarthritic pathogenic process. We then investigated if we could block the BCP-IL6 loop by either targeting IL-6 production or BCP crystal deposits. Treatment of chondrocytes with anti-IL-6 antibodies or inhibitors of IL-6- signaling pathway significantly inhibited IL-6-induced crystal formation. Similarly, sodium thiosulfate (STS), a well-known systemic calcification inhibitor, decreased crystal deposition as well as HA-induced IL-6 secretion in chondrocytes and, in vivo, it decreased crystal deposits size and cartilage erosion in menisectomized knees. Interestingly, we also found that xanthine-oxidoreductase (XO) inhibitors inhibited both IL-6 production and calcium crystal depositis in chondrocytes. We began to unravel the mechanisms involved in this coordinate modulation of IL-6 and mineralization. STS inhibited Reactive Oxygen Species (ROS) generation and we are currently investigating whether XO represents a major source of ROS in chondrocyte mineralization. Finally, we ruled out that IL-1 activation/signaling plays a role in the murine model of OA induced by menisectomy, as IL-1a/ß, the IL-1 R associated molecule MyD88 and NLRP3 inflammasome deficient mice were not protected in this model of OA. Moreover TLR-1, -2, -4,-6 deficient mice had a phenotype similar to that of wild-type mice. Altogether our results demonstrated a self-amplification loop between BCP crystals deposition and IL-6 production, which represents an aggravating process in OA pathogenesis. As currently prescribed OA drugs are addressing OA symptoms,our results highlight a potential novel treatment strategy whereby inhibitors of calcium- containing crystal formation and IL-6 could be combined to form the basis of a disease modifying treatment and alter the course of OA.

Oxidative stress in aging: advances in proteomic approaches

Aging is a gradual, complex process in which cells, tissues, organs, and the whole organism itself deteriorate in a progressive and irreversible manner that, in the majority of cases, implies pathological conditions that affect the individual"s Quality of Life (QOL). Although extensive research efforts in recent years have been made, the anticipation of aging and prophylactic or treatment strategies continue to experience major limitations. In this review, the focus is essentially on the compilation of the advances generated by cellular expression profile analysis through proteomics studies (two-dimensional [2D] electrophoresis and mass spectrometry [MS]), which are currently used as an integral approach to study the aging process. Additionally, the relevance of the oxidative stress factors is discussed. Emphasis is placed on postmitotic tissues, such as neuronal, muscular, and red blood cells, which appear to be those most frequently studied with respect to aging. Additionally, models for the study of aging are discussed in a number of organisms, such as Caenorhabditis elegans, senescence-accelerated probe-8 mice (SAMP8), naked mole-rat (Heterocephalus glaber), and the beagle canine. Proteomic studies in specific tissues and organisms have revealed the extensive involvement of reactive oxygen species (ROS) and oxidative stress in aging.