Mechanisms underlying cytotoxicity induced by engineered nanomaterials: a review of in vitro studies

Engineered nanomaterials are emerging functional materials with technologically interesting properties and a wide range of promising applications, such as drug delivery devices, medical imaging and diagnostics, and various other industrial products. However, concerns have been expressed about the risks of such materials and whether they can cause adverse effects. Studies of the potential hazards of nanomaterials have been widely performed using cell models and a range of in vitro approaches. In the present review, we provide a comprehensive and critical literature overview on current in vitro toxicity test methods that have been applied to determine the mechanisms underlying the cytotoxic effects induced by the nanostructures. The small size, surface charge, hydrophobicity and high adsorption capacity of nanomaterial allow for specific interactions within cell membrane and subcellular organelles, which in turn could lead to cytotoxicity through a range of different mechanisms. Finally, aggregating the given information on the relationships of nanomaterial cytotoxic responses with an understanding of its structure and physicochemical properties may promote the design of biologically safe nanostructures.

Ewing sarcoma cancer stem cells : from mechanisms to therapeutic targeting

Le sarcome d'Ewing (SE) est la 2ème tumeur des os la plus fréquente chez les enfants, et le pronostic est sombre au stade métastatique. La pathogenèse du SE repose sur une translocation, provocant la fusion du domaine activateur du facteur de transcription EWS, avec la partie liant l'ADN de la protéine FLI-1. Les cellules souches cancéreuses (CSC) sont supposées être les moteurs de la croissance tumorale, et représente de ce fait des cibles thérapeutiques préférentielles. Dans ce travail nous nous sommes efforcés de comprendre, ainsi que de cibler les mécanismes liés à l'émergence des CSC dans le sarcome d'Ewing. La formation des CSC du ES est liée à un défaut de maturation des miRNAs provoqué par une sous-expression d'un gène, TARBP2, dans les CSC. Ce défaut de maturation peut être corrigé par un traitement des cellules avec de l'enoxacine, une fluoroquinolone utilisée pour traiter les infections urinaires. L'enoxacine seule n'étant pas suffisante pour éradiquer les tumeurs in vivo, nous avons testé la combinaison d'une thérapie ciblée sur les CSC avec une chimiothérapie classique, la doxorubicine, ciblant les cellules différentiées. In vitro l'enoxacine induit l'apoptose dans les CCS sans affecter les cellules différentiées, alors que à l'inverse, la doxorubicine n'affecte que les cellules de la « masse » tumorale. In vivo la combinaison de ces deux drogues inhibe la croissance de tumeurs provenant de cellules primaires xenotranplantées et éradique les CSCs. Nos résultats mettent en lumière une nouvelle approche thérapeutique directement applicable pour le sarcome d'Ewing, et pourraient ainsi rapidement déboucher sur des essais cliniques. Dans la deuxième partie de ce travail nous avons essayé de comprendre comment EWS-FLI1, la protéine de fusion issue de la translocation chromosomique du sarcome d'Ewing conduit à la génération des CSC. Pour cela nous avons effectué des ChIPseq (immunoprecipitation de la chromatine suivi de séquençage) pour EWS-FLI1 ainsi que pour certaines modifications histoniques. -- Ewing sarcoma family tumors (ESFT) are the second most frequent bone tumors in children and have a high rate of recurrence when metastatic at presentation. The pathogenesis of Ewing sarcoma is underlayed by a translocation, leading to the fusion of the trans-activating domain of EWS with the FLU DNA binding domain. Cancer stem cells (CSCs) are thought to be the driving force of tumor growth. In this work we focused on understanding the mechanisms underlying ESFT CSC emergence as well as defining targeted therapeutic strategies. Emergence of CSCs in ESFT has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. As enoxacin alone is not sufficient to reverse tumor growth in vivo, we assessed the effect of combining a drug that abrogates CSC properties with doxorubicin, a standard-of-care therapy in ESFT. Primary ESFT CSCs and bulk tumor cells were treated with different concentration of drugs and displayed divergent responses to doxorubicin and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin induced apoptosis but only in ESFT spheres and specifically on the CD133+ population. In combination, the two drugs markedly depleted CSC and strongly reduced primary growth in xenograft assays of two primary ESFT. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation. In the second part of this work we performed chromatin immunopercipitation on CSCs and bulk cells for EWS-FLI1 binding as well as some chromatin modifications, and concluded that EWS-FLI1 shows cell context dependent binding.

Manipulation of arthropod sex determination by endosymbionts: diversity and molecular mechanisms.

Arthropods exhibit a large variety of sex determination systems both at the chromosomal and molecular level. Male heterogamety, female heterogamety, and haplodiploidy occur frequently, but partially different genes are involved. Endosymbionts, such as Wolbachia, Cardinium,Rickettsia, and Spiroplasma, can manipulate host reproduction and sex determination. Four major reproductive manipulation types are distinguished: cytoplasmic incompatibility, thelytokous parthenogenesis, male killing, and feminization. In this review, the effects of these manipulation types and how they interfere with arthropod sex determination in terms of host developmental timing, alteration of sex determination, and modification of sexual differentiation pathways are summarized. Transitions between different manipulation types occur frequently which suggests that they are based on similar molecular processes. It is also discussed how mechanisms of reproductive manipulation and host sex determination can be informative on each other, with a special focus on haplodiploidy. Future directions on how the study of endosymbiotic manipulation of host reproduction can be key to further studies of arthropod sex determination are shown.

The alpha1-adrenergic receptors in cardiac hypertrophy: Signaling mechanisms and functional implications.

Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α1-adrenergic receptors (α1-ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α1-AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α1-adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α1-AR in the heart and their functional implications in cardiac hypertrophy.

Mechanisms of Antifungal Drug Resistance.

Antifungal therapy is a central component of patient management for acute and chronic mycoses. Yet, treatment choices are restricted because of the sparse number of antifungal drug classes. Clinical management of fungal diseases is further compromised by the emergence of antifungal drug resistance, which eliminates available drug classes as treatment options. Once considered a rare occurrence, antifungal drug resistance is on the rise in many high-risk medical centers. Most concerning is the evolution of multidrug- resistant organisms refractory to several different classes of antifungal agents, especially among common Candida species. The mechanisms responsible are mostly shared by both resistant strains displaying inherently reduced susceptibility and those acquiring resistance during therapy. The molecular mechanisms include altered drug affinity and target abundance, reduced intracellular drug levels caused by efflux pumps, and formation of biofilms. New insights into genetic factors regulating these mechanisms, as well as cellular factors important for stress adaptation, provide a foundation to better understand the emergence of antifungal drug resistance.

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.

Mechanisms of OGT-mediated HCF-1 protein maturation

Post-translational protein modifications are crucial for many fundamental cellular and extracellular processes and greatly contribute to the complexity of organisms. Human HCF-1 is a transcriptional co-regulator that undergoes complex protein maturation involving reversible and irreversible post-translational modifications. Upon synthesis as a large precursor protein, HCF-1 undergoes extensive reversible glycosylation with β-N-acetylglucosamine giving rise to O-linked-β-N-acetylglucosamine (O-GlcNAc) modified serines and threonines. HCF-1 also undergoes irreversible site-specific proteolysis, which is important for one of HCF-1's major functions - the regulation of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by a single enzyme with an unusual dual enzymatic activity, the O-GlcNAc transferase (OGT). HCF-1 is cleaved by OGT at any of six highly conserved 26 amino acid repeated sequences (HCF-1PRO repeats), but the mechanisms and the substrate requirements for OGT-mediated cleavage are not understood. In the present work, I characterized substrate requirements for OGT-mediated cleavage and O-GlcNAcylation of HCF-1. I identified key elements within the HCF-1PRO-repeat sequence that are important for proteolysis. Remarkably, an invariant single amino acid side-chain within the HCF-1PRO-repeat sequence displays particular OGT-binding properties and is essential for proteolysis. Additionally, I characterized substrate requirements for proteolysis outside of the HCF-1PRO repeat and identified a novel, highly O-GlcNAcylated OGT-binding sequence that enhances cleavage of the first HCF-1PRO repeat. These results link OGT association and its O-GlcNAcylation activities to HCF-1PRO-repeat proteolysis.

Fetal programming and epigenetic mechanisms in arterial hypertension.

PURPOSE OF REVIEW: To provide an overview of available evidence of the potential role of epigenetics in the pathogenesis of hypertension and vascular dysfunction. RECENT FINDINGS: Arterial hypertension is a highly heritable condition. Surprisingly, however, genetic variants only explain a tiny fraction of the phenotypic variation and the term 'missing heritability' has been coined to describe this phenomenon. Recent evidence suggests that phenotypic alteration that is unrelated to changes in DNA sequence (thereby escaping detection by classic genetic methodology) offers a potential explanation. Here, we present some basic information on epigenetics and review recent work consistent with the hypothesis of epigenetically induced arterial hypertension. SUMMARY: New technologies that enable the rigorous assessment of epigenetic changes and their phenotypic consequences may provide the basis for explaining the missing heritability of arterial hypertension and offer new possibilities for treatment and/or prevention.

Synaptic Integration of Adult-Born Hippocampal Neurons Is Locally Controlled by Astrocytes.

Adult neurogenesis is regulated by the neurogenic niche, through mechanisms that remain poorly defined. Here, we investigated whether niche-constituting astrocytes influence the maturation of adult-born hippocampal neurons using two independent transgenic approaches to block vesicular release from astrocytes. In these models, adult-born neurons but not mature neurons showed reduced glutamatergic synaptic input and dendritic spine density that was accompanied with lower functional integration and cell survival. By taking advantage of the mosaic expression of transgenes in astrocytes, we found that spine density was reduced exclusively in segments intersecting blocked astrocytes, revealing an extrinsic, local control of spine formation. Defects in NMDA receptor (NMDAR)-mediated synaptic transmission and dendrite maturation were partially restored by exogenous D-serine, whose extracellular level was decreased in transgenic models. Together, these results reveal a critical role for adult astrocytes in local dendritic spine maturation, which is necessary for the NMDAR-dependent functional integration of newborn neurons.

Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake

Previous studies have shown that rat intestinal immunoglobulin A (IgA) concentration and lymphocyte composition of the intestinal immune system were influenced by a highly enriched cocoa diet. The aim of this study was to dissect the mechanisms by which a long-term high cocoa intake was capable of modifying gut secretory IgA in Wistar rats. After 7 weeks of nutritional intervention, Peyer's patches, mesenteric lymph nodes and the small intestine were excised for gene expression assessment of IgA, transforming growth factor ß, C-C chemokine receptor-9 (CCR9), interleukin (IL)-6, CD40, retinoic acid receptors (RAR¿ and RARß), C-C chemokine ligand (CCL)-25 and CCL28 chemokines, polymeric immunoglobulin receptor and toll-like receptors (TLR) expression by real-time polymerase chain reaction. As in previous studies, secretory IgA concentration decreased in intestinal wash and fecal samples after cocoa intake. Results from the gene expression showed that cocoa intake reduced IgA and IL¿6 in Peyer's patches and mesenteric lymph nodes, whereas in small intestine, cocoa decreased IgA, CCR9, CCL28, RAR¿ and RARß. Moreover, cocoa-fed animals presented an altered TLR expression pattern in the three compartments studied. In conclusion, a high-cocoa diet down-regulated cytokines such as IL-6, which is required for the activation of B cells to become IgA-secreting cells, chemokines and chemokine receptors, such as CCL28 and CCR9 together with RAR¿ and RARß, which are involved in the gut homing of IgA-secreting cells. Moreover, cocoa modified the cross-talk between microbiota and intestinal cells as was detected by an altered TLR pattern. These overall effects in the intestine may explain the intestinal IgA down-regulatory effect after the consumption of a long-term cocoa-enriched diet.

Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer.

Proteases are important for regulating multiple tumorigenic processes, including angiogenesis, tumor growth, and invasion. Elevated protease expression is associated with poor patient prognosis across numerous tumor types. Several multigene protease families have been implicated in cancer, including cysteine cathepsins. However, whether individual family members have unique roles or are functionally redundant remains poorly understood. Here we demonstrate stage-dependent effects of simultaneously deleting cathepsin B (CtsB) and CtsS in a murine pancreatic neuroendocrine tumor model. Early in tumorigenesis, the double knockout results in an additive reduction in angiogenic switching, whereas at late stages, several tumorigenic phenotypes are unexpectedly restored to wild-type levels. We identified CtsZ, which is predominantly supplied by tumor-associated macrophages, as the compensatory protease that regulates the acquired tumor-promoting functions of lesions deficient in both CtsB and CtsS. Thus, deletion of multiple cathepsins can lead to stage-dependent, compensatory mechanisms in the tumor microenvironment, which has potential implications for the clinical consideration of selective versus pan-family cathepsin inhibitors in cancer.

Interleukin-6 and chondrocyte mineralisation act in tandem to promote experimental osteoarthritis.

OBJECTIVES: Basic calcium phosphate (BCP) crystal and interleukin 6 (IL-6) have been implicated in osteoarthritis (OA). We hypothesise that these two factors may be linked in a reciprocal amplification loop which leads to OA. METHODS: Primary murine chondrocytes and human cartilage explants were incubated with hydroxyapatite (HA) crystals, a form of BCP, and the modulation of cytokines and matrix-degrading enzymes assayed. The ability of IL-6 to stimulate chondrocyte calcification was assessed in vitro. The mechanisms underlying the effects of HA on chondrocytes were investigated using chemical inhibitors, and the pathways mediating IL-6-induced calcification characterised by quantifying the expression of genes involved in chondrocyte mineralisation. The role of calcification in vivo was studied in the meniscectomy model of murine OA (MNX), and the link between IL-6 and cartilage degradation investigated by histology. RESULTS: In chondrocytes, BCP crystals stimulated IL-6 secretion, further amplified in an autocrine loop, through signalling pathways involving Syk and PI3 kinases, Jak2 and Stat3 molecules. Exogenous IL-6 promoted calcium-containing crystal formation and upregulation of genes involved in calcification: the pyrophosphate channel Ank, the calcium channel Annexin5 and the sodium/phosphate cotransporter Pit-1. Treatment of chondrocytes with IL-6 inhibitors significantly inhibited IL-6-induced crystal formation. In meniscectomised mice, increasing deposits of BCP crystals were observed around the joint and correlated with cartilage degradation and IL-6 expression. Finally, BCP crystals induced proteoglycan loss and IL-6 expression in human cartilage explants, which were reduced by an IL-6 inhibitor. CONCLUSIONS: BCP crystals and IL-6 form a positive feedback loop leading to OA. Targeting calcium-containing crystal formation and/or IL-6 are promising therapeutic strategies in OA.

The biological embedding of social differences in ageing trajectories.

The occurrence of adult disease is related to lifetime experiences and, at least in part, to early life events. It is now well established that socioeconomic circumstances across the lifetime are major determinants of adult health and disease, and the current economic crisis is amplifying susceptibility to disease and unhealthy ageing in disadvantaged subgroups of the population. In adulthood, the gap between social groups is extensive in terms of mortality, functional performances and cognitive capacity. Since the occurrence of adult disease is related to lifetime experiences, including early life exposures, late-life preventive efforts may be of limited efficacy, particularly in disadvantaged subgroups. We now have the analytical tools to understand mechanisms that underlie life-long susceptibility to unhealthy ageing, and new knowledge can lead to better and more effective mechanisms to prevent diseases and reduce health inequalities. In this perspective, we first discuss the impact of recent changes in the understanding of chronic disease aetiology on our interpretation of the influence of life-course socioeconomic status (SES) on health and ageing. We then propose a model for integrating the exposome concept (the myriad of exposures derived from exogenous and endogenous sources) into the analysis of life-course socioeconomic differentials in ageing.

Cerebral Lactate Metabolism After Traumatic Brain Injury.

Cerebral energy dysfunction has emerged as an important determinant of prognosis following traumatic brain injury (TBI). A number of studies using cerebral microdialysis, positron emission tomography, and jugular bulb oximetry to explore cerebral metabolism in patients with TBI have demonstrated a critical decrease in the availability of the main energy substrate of brain cells (i.e., glucose). Energy dysfunction induces adaptations of cerebral metabolism that include the utilization of alternative energy resources that the brain constitutively has, such as lactate. Two decades of experimental and human investigations have convincingly shown that lactate stands as a major actor of cerebral metabolism. Glutamate-induced activation of glycolysis stimulates lactate production from glucose in astrocytes, with subsequent lactate transfer to neurons (astrocyte-neuron lactate shuttle). Lactate is not only used as an extra energy substrate but also acts as a signaling molecule and regulator of systemic and brain glucose use in the cerebral circulation. In animal models of brain injury (e.g., TBI, stroke), supplementation with exogenous lactate exerts significant neuroprotection. Here, we summarize the main clinical studies showing the pivotal role of lactate and cerebral lactate metabolism after TBI. We also review pilot interventional studies that examined exogenous lactate supplementation in patients with TBI and found hypertonic lactate infusions had several beneficial properties on the injured brain, including decrease of brain edema, improvement of neuroenergetics via a "cerebral glucose-sparing effect," and increase of cerebral blood flow. Hypertonic lactate represents a promising area of therapeutic investigation; however, larger studies are needed to further examine mechanisms of action and impact on outcome.