Interaction of cationic ultrasmall superparamagnetic iron oxide nanoparticles with human melanoma cells.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) are currently under development for the intracellular delivery of therapeutics. However, the mechanisms of cellular uptake and the cellular reaction to this uptake, independent of therapeutics, are not well defined. The interactions of biocompatible cationic aminoUSPIONs with human cells was studied in 2D and 3D cultures using biochemical and electron microscopy techniques. AminoUSPIONs were internalized by human melanoma cells in 2D and 3D cultures. Uptake was clathrin mediated and the particles localized in lysosomes, inducing activation of the lysosomal cathepsin D and decreasing the expression of the transferrin receptor in human melanoma cells and/or skin fibroblasts. AminoUSPIONs deeply invaded 3D spheroids of human melanoma cells. Thus, aminoUSPIONs can invade tumors and their uptake by human cells induces cell reaction.

The Candida albicans plasma membrane protein Rch1p, a member of the vertebrate SLC10 carrier family, is a novel regulator of cytosolic Ca(2+) homoeostasis.

Candida albicans RCH1 (regulator of Ca(2+) homoeostasis 1) encodes a protein of ten TM (transmembrane) domains, homologous with human SLC10A7 (solute carrier family 10 member 7), and Rch1p localizes in the plasma membrane. Deletion of RCH1 confers hypersensitivity to high concentrations of extracellular Ca(2+) and tolerance to azoles and Li(+), which phenocopies the deletion of CaPMC1 (C. albicans PMC1) encoding the vacuolar Ca(2+) pump. Additive to CaPMC1 mutation, lack of RCH1 alone shows an increase in Ca(2+) sensitivity, Ca(2+) uptake and cytosolic Ca(2+) level. The Ca(2+) hypersensitivity is abolished by cyclosporin A and magnesium. In addition, deletion of RCH1 elevates the expression of CaUTR2 (C. albicans UTR2), a downstream target of the Ca(2+)/calcineurin signalling. Mutational and functional analysis indicates that the Rch1p TM8 domain, but not the TM9 and TM10 domains, are required for its protein stability, cellular functions and subcellular localization. Therefore Rch1p is a novel regulator of cytosolic Ca(2+) homoeostasis, which expands the functional spectrum of the vertebrate SLC10 family.

A missing sugar prevents glucose entry: a new twist on insulin secretion.

The signaling pathway that regulates glucose-stimulated insulin secretion depends on glucose metabolism, which is itself controlled by glucokinase. In a recent issue of Cell, show that altering N-glycosylation of the GLUT2 glucose transporter prevents its anchoring and retention at the cell surface; this impairs glucose uptake and insulin secretion.

Antigen-secretory IgA immune complexes are retro-transported into mouse Peyer's patches: immunotargeting to dendritic cells

RÉSUMÉ Les plaques de Peyer (PP) représentent le site d'entrée majeur des pathogènes au niveau des muqueuses intestinales. Après avoir traversé la cellule M, l'antigène est pris en charge par les cellules dendritiques (DC) de la région sub-épithéliale du dôme des PP. Ces dernières activent une réponse immunitaire qui conduit à la production de l'IgA de sécrétion (SIgA), l'anticorps majeur au niveau muqueux. Des études précédentes dans notre laboratoire ont démontré qu'après administration de SIgA dans des anses intestinales de souris, les SIgA se lient spécifiquement aux cellules M, entrent dans les PP, et sont éventuellement internalisées par les DC. Le but de ce travail est de comprendre la relevance biologique de l'entrée des SIgA dans les PP et leur relevance physiologique dans l'homéostasie mucosale. Dans un premier temps, nous avons montré en utilisant une méthode de purification optimisée basée sur une isolation magnétique, que, en plus des DC myéloïdes (CD11c+/CD11b+) et des DC lymphoïdes (CD11c+/CD8+), les PP de souris contiennent un nouveau sous-type de DC exprimant les marqueurs CD11c et CD19. L'utilisation de la microscopie confocale nous a permis de démontrer que les DC myéloïdes internalisent des SIgA, contrairement aux DC lymphoïdes qui n'interagissent pas avec les SIgA, alors que le nouveau sous-type de DC exprimant CD19 lie les SIgA. En plus, nous avons démontré qu'aucune des DC de rate, de ganglion bronchique ou de ganglion inguinal interagit avec les SIgA. Dans le but d'explorer si les SIgA peuvent délivrer des antigènes aux DC des PP in vivo, nous avons administré des complexes immunitaires formés de Shigella flexneri complexées à des SIgA, dans des anses intestinales de souris. Nous avons observé une entrée dans les PP, suivie d'une migration vers les ganglions mésentériques drainants, contrairement aux Shigella flexneri seules, qui n'infectent pas la souris par la voie intestinale. Shigella flexneri délivrée par SIgA n'induit pas de destruction tissulaire au niveau de l'intestin. En plus de l'exclusion immunitaire, ces résultats suggèrent un nouveau rôle des SIgA, qui consiste à transporter des antigènes à l'intérieur des PP dans un contexte non-inflammatoire. RÉSUMÉ DESTINÉ À UN LARGE PUBLIC L'intestin a pour rôle principal d'absorber les nutriments digérés tout au long du tube digestif, et de les faire passer dans le compartiment intérieur sanguin. Du fait de son exposition chronique avec un monde extérieur constitué d'aliments et de bactéries, l'intestin est un endroit susceptible aux infections et a donc besoin d'empêcher l'entrée de microbes. Pour cela, l'intestin est tapissé de "casernes" appelées les plaques de Peyer, qui appartiennent à un système de défense appelé système immunitaire muqueux. Les plaques de Peyer sont composées de différents types de cellules, ayant pour rôle de contrôler l'entrée de microbes et de développer une réaction immunitaire lors d'infection. Cette réaction immunitaire contre les microbes (antigènes) débute par la prise en charge de l'antigène par des sentinelles, les cellules dendritiques. L'antigène est préparé de façon à être reconnu par d'autres cellules appelées lymphocytes T capables d'activer d'autres cellules, les lymphocytes B. La réaction immunitaire résulte dans la production par les lymphocytes B d'un anticorps spécifique appelé IgA de sécrétion (SIgA) au niveau de la lumière intestinale. De manière classique, le rôle de SIgA au niveau de la lumière intestinale consiste à enrober les microbes et donc exclure leur entrée dans le compartiment intérieur. Dans ce travail, nous avons découvert une nouvelle fonction des SIgA qui consiste à introduire des antigènes dans les plaques de Peyer, et de les diriger vers les cellules dendritiques. Sachant que les SIgA sont des anticorps qui ne déclenchent pas de réactions de défense violentes dites inflammatoires, l'entrée des antigènes via SIgA serait en faveur d'une défense intestinale maîtrisée sans qu'il y ait d'inflammation délétère. Ces résultats nous laissent supposer que l'entrée d'antigènes via SIgA pourrait conduire le système immunitaire muqueux à reconnaître ces antigènes de manière appropriée. Ce mécanisme pourrait expliquer les désordres immunitaires de types allergiques et maladies auto-immunitaires que l'on rencontre chez certaines personnes déficientes en IgA, chez qui cette lecture d'antigènes de manière correcte serait inadéquate. ABSTRACT Peyer's patches (PP) represent the primary site for uptake and presentation of ingested antigens in the intestine. Antigens are sampled by M cells, which pass them to underlying antigen-presenting cells including dendritic cells (DC). This leads to the induction of mucosal T cell response that is important for the production of secretory IgA (SIgA), the chief antibody at mucosal surfaces. Previous studies in the laboratory have shown that exogenous SIgA administrated into mouse intestinal loop binds specifically to M cells, enter into PP, and is eventually internalized by DC. The aim of this work is to understand the biological significance of the SIgA uptake by PP DC and its physiological relevance for mucosal homeostasis. As a first step, we have shown by using an optimized MACS method that, in addition to the CD11c+/CD11b+ (myeloid DC) and CD11c+/CD8+ (lymphoid DC) subtypes, mouse PP contain a novel DC subtype exhibiting both CD11c and CD19 markers. By using a combination of MACS isolation and confocal microscopy, we have demonstrated that in contrast to the lymphoid DC which do not interact with SIgA, the myeloid DC internalize SIgA, while the CD19+ subtype binds SIgA on its surface. Neither spleen DC, nor bronchial-lymph node DC, nor inguinal lymph node DC exhibit such a binding specificity. To test whether SIgA could deliver antigens to PP DC in vivo, we administered SIgA-Shigella flexneri immune complexes into mouse intestinal loop containing a PP. We found that (i) SIgA-Shigella flexneri immune complexes enter the PP and are internalized by sub-epithelial dome PP DC, in contrast to Shigella flexneri alone that does not penetrate the intestinal epithelia in mice, (ii) immune complexes migrate to the draining mesenteric lymph node, (iii) Shigella flexneri carried via SIgA do not induce intestinal tissue destruction. Our results suggest that in addition to immune exclusion, SIgA transports antigens back to the PP under non-inflammatory conditions.

Evidence for tuning adipocytes ICER levels for obesity care.

Abnormal adipokine production, along with defective uptake and metabolism of glucose within adipocytes, contributes to insulin resistance and altered glucose homeostasis. Recent research has highlighted one of the mechanisms that accounts for impaired production of adiponectin (ADIPOQ) and adipocyte glucose uptake in obesity. In adipocytes of human obese subjects and mice fed with a high fat diet, the level of the inducible cAMP early repressor (ICER) is diminished. Reduction of ICER elevates the cAMP response element binding protein (CREB) activity, which in turn increases the repressor activating transcription factor 3. In fine, the cascade triggers reduction in the ADIPOQ and GLUT4 levels, which ultimately hampers insulin-mediated glucose uptake. The c-Jun N-terminal kinase (JNK) interacting-protein 1, also called islet brain 1 (IB1), is a target of CREB/ICER that promotes JNK-mediated insulin resistance in adipocytes. A rise in IB1 and c-Jun levels accompanies the drop of ICER in white adipose tissues of obese mice when compared with mice fed with a chow diet. Other than the expression of ADIPOQ and glucose transport, decline in ICER expression might impact insulin signaling. Impairment of ICER is a critical issue that will need major consideration in future therapeutic purposes.

Assessing ageing of individual T lymphocytes: Mission impossible?

Effector T lymphocytes are the progeny of a limited number of antigen- specific precursor cells and it has been estimated that clonotypic human T cells may expand million fold on their way reaching high cell numbers that are sufficient for immune protection. Moreover, memory T cell responses are characterized by repetitive expansion of antigen-specific T cell clonotypes, and limitations in the proliferative capacity could lead to immune senescence. Because telomeres progressively shorten as a function of cell division, telomere length is a powerful indicator of the replicative in vivo history of human T lymphocytes. In this review, we summarize observations made over the last decade on telomere length dynamics of well-defined T cell populations derived from healthy donors and patients with infectious disease or cancer. We focus on T cell differentiation, T cell ageing, and natural and vaccine induced immune responses. We also discuss the scientific evidence for in vivo replicative senescence of antigen-specific T cells, and evaluate the available methods for measuring telomere lengths and telomerase activity, and their potential and limitations to increase our understanding of T cell physiology. (C) 2007 Elsevier Ireland Ltd. All rights reserved.

Adipose tissue integrity as a prerequisite for systemic energy balance: a critical role for peroxisome proliferator-activated receptor gamma.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is an essential regulator of adipocyte differentiation, maintenance, and survival. Deregulations of its functions are associated with metabolic diseases. We show here that deletion of one PPARgamma allele not only affected lipid storage but, more surprisingly, also the expression of genes involved in glucose uptake and utilization, the pentose phosphate pathway, fatty acid synthesis, lipolysis, and glycerol export as well as in IR/IGF-1 signaling. These deregulations led to reduced circulating adiponectin levels and an energy crisis in the WAT, reflected in a decrease to nearly half of its intracellular ATP content. In addition, there was a decrease in the metabolic rate and physical activity of the PPARgamma(+/-) mice, which was abolished by thiazolidinedione treatment, thereby linking regulation of the metabolic rate and physical activity to PPARgamma. It is likely that the PPARgamma(+/-) phenotype was due to the observed WAT dysfunction, since the gene expression profiles associated with metabolic pathways were not affected either in the liver or the skeletal muscle. These findings highlight novel roles of PPARgamma in the adipose tissue and underscore the multifaceted action of this receptor in the functional fine tuning of a tissue that is crucial for maintaining the organism in good health.

Lead acetate toxicity in vitro: Dependence on the cell composition of the cultures.

It is well known that exposure to low doses of lead causes long-lasting neurobehavioural deficits, but the cellular changes underlying these behavioural changes remain to be elucidated. A protective role of glial cells on neurons through lead sequestration by astrocytes has been proposed. The possible modulation of lead neurotoxicity by neuron-glia interactions was examined in three-dimensional cultures of foetal rat telencephalon. Mixed-brain cell cultures or cultures enriched in either neurons or glial cells were treated for 10 days with lead acetate (10(-6) m), a concentration below the limit of cytotoxicity. Intracellular lead content and cell type-specific enzyme activities were determined. It was found that in enriched cultures neurons stored more lead than glial cells, and each cell type alone stored more lead than in co-culture. Moreover, glial cells but not neurons were more affected by lead in enriched culture than in co-culture. These results show that neuron-glia interactions attenuate the cellular lead uptake and the glial susceptibility to lead, but they do not support the idea of a protective role of astrocytes.

A novel protein family mediates Casparian strip formation in the endodermis.

Polarized epithelia are fundamental to multicellular life. In animal epithelia, conserved junctional complexes establish membrane diffusion barriers, cellular adherence and sealing of the extracellular space. Plant cellular barriers are of independent evolutionary origin. The root endodermis strongly resembles a polarized epithelium and functions in nutrient uptake and stress resistance. Its defining features are the Casparian strips, belts of specialized cell wall material that generate an extracellular diffusion barrier. The mechanisms localizing Casparian strips are unknown. Here we identify and characterize a family of transmembrane proteins of previously unknown function. These 'CASPs' (Casparian strip membrane domain proteins) specifically mark a membrane domain that predicts the formation of Casparian strips. CASP1 displays numerous features required for a constituent of a plant junctional complex: it forms complexes with other CASPs; it becomes immobile upon localization; and it sediments like a large polymer. CASP double mutants display disorganized Casparian strips, demonstrating a role for CASPs in structuring and localizing this cell wall modification. To our knowledge, CASPs are the first molecular factors that are shown to establish a plasma membrane and extracellular diffusion barrier in plants, and represent a novel way of epithelial barrier formation in eukaryotes.

Alteration of glucose metabolism in cultured astrocytes after AQP9-small interference RNA application.

Aquaglyceroporin-9 (AQP9) facilitates diffusion of water and energy substrates such as glycerol and monocarboxylates. AQP9 is present in plasma membrane and mitochondria of astrocytes and catecholaminergic neurons, suggesting that it plays a role in the energetic status of these cells. Using specific small interference RNA directed against AQP9 in astrocyte cultures, we showed that glycerol uptake is decreased which is associated with an increase in glucose uptake and oxidative metabolism. Our results not only confirm the presence of AQP9 in astrocytes but also suggest that changes in AQP9 expression alter glial energy metabolism.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro.

BACKGROUND: Plasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA). RESULTS: At first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68-75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NPâeuro0/00>âeuro0/00chitosan DNA NPâeuro0/00=âeuro0/00DNA unloaded chitosan NPâeuro0/00>âeuro0/00control (culture medium). CONCLUSIONS: Chitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

Lead acetate toxicity in vitro: Dependence on the cell composition of the cultures.

It is well known that exposure to low doses of lead causes long-lasting neurobehavioural deficits, but the cellular changes underlying these behavioural changes remain to be elucidated. A protective role of glial cells on neurons through lead sequestration by astrocytes has been proposed. The possible modulation of lead neurotoxicity by neuron-glia interactions was examined in three-dimensional cultures of foetal rat telencephalon. Mixed-brain cell cultures or cultures enriched in either neurons or glial cells were treated for 10 days with lead acetate (10(-6) m), a concentration below the limit of cytotoxicity. Intracellular lead content and cell type-specific enzyme activities were determined. It was found that in enriched cultures neurons stored more lead than glial cells, and each cell type alone stored more lead than in co-culture. Moreover, glial cells but not neurons were more affected by lead in enriched culture than in co-culture. These results show that neuron-glia interactions attenuate the cellular lead uptake and the glial susceptibility to lead, but they do not support the idea of a protective role of astrocytes.

Availability of a soluble phosphorus source applied to soil samples with different acidicity levels

Considerations on the interactions of P in the soil-plant system have a long history, but are still topical and not yet satisfactorily understood. One concern is the effect of liming before or after application of soluble sources on the crop yield and efficiency of available P under these conditions. The aim of this study was to evaluate the effect of soil acidity on availability of P from a soluble source, based on plant growth and chemical extractants. Nine soil samples were incubated with a dose of 200 mg kg-1 P in soil with different levels of previously adjusted acidity (pH H2O 4.5; 5.0; 5.5; 6.0 and 6.5) and compared to soils without P application. After 40 days of soil incubation with a P source, each treatment was limed again so that all pH values were adjusted to 6.5 and then sorghum was planted. After the first and second liming the P levels were determined by the extractants Mehlich-1, Bray-1 and Resin, and the fractionated inorganic P forms. In general, the different acidity levels did not influence the P availability measured by plant growth and P uptake at the studied P dose. For some soils however these values increased or decreased according to the initial soil pH (from 4.5 to 6.5). Plant growth, P uptake and P extractable by Mehlich-1 and Bray-1 were significantly correlated, unlike resin-extractable P, at pH values raised to 6.5. These latter correlations were however significant before the second liming. The P contents extracted by Mehlich-1 and Bray-1 were significantly correlated with each other in the entire test range of soil acidity, even after adjusting pH to 6.5, besides depending on the soil buffering capacity for P. Resin was also sensitive to the properties that express the soil buffering capacity for P, but less clearly than Mehlich-1 and Bray-1. The application of triple superphosphate tended to increase the levels of P-Al, P-Fe and P-Ca and the highest P levels extracted by Bray-1 were due to a higher occurrence of P-Al and P-Fe in the soils.

Cation dynamics in soils with different salinity levels growing irrigated rice

Salinity levels in soils of the Outer Coastal Plain of Rio Grande do Sul, Brazil, can be high, due to excess of Na in the irrigation water, evapotranspiration and soil development from marine sediments. The cultivation of irrigated rice could be an alternative, since ion uptake as well as leaching by the establishment of a water layer could mitigate the effects of soil salinity. This study aimed to evaluate the dynamics of basic cations in the solution of Albaqualf soils with different salinity levels growing irrigated rice. The plow layer contained exchangeable Na percentages (ESP) of 5.6, 9.0, 21.2 and 32.7 %. The plant stand, dry matter, Na, K and Ca + Mg uptake at full flowering and grain yield were evaluated. The levels of Na, K, Ca + Mg and electrical conductivity (EC) in the soil solution were also measured weekly during the rice cycle at four soil depths, in the water layer and irrigation water. The Na, K and Ca + Mg uptake by rice at full flowering was used to estimate ion depletion from the layer under root influence. Soil salinity induced a reduction in the rice stand, especially in the soil with ESP of 32.7 %, resulting in lower cation uptake and very low yield at that site. As observed in the water layer and irrigation water, the Na, K, Ca + Mg and EC levels in the soil solution decreased with time at depths of 5, 10 and 20 cm, regardless of the original soil salinity, showing that cation dynamics in the plow layer was determined by leaching and root uptake, rather than by the effect of evapoconcentration of basic cations in the soil surface layer.