Targeting antigen in mature dendritic cells for simultaneous stimulation of CD4+ and CD8+ T cells

Due to their potent immunostimulatory capacity, dendritic cells (DC) have become the centerpiece of many vaccine regimens. Immature DC (DCimm) capture, process, and present Ags to CD4(+) lymphocytes, which reciprocally activate DCimm through CD40, and the resulting mature DC (DCmat) loose phagocytic capacity, but acquire the ability to efficiently stimulate CD8(+) lymphocytes. Recombinant vaccinia viruses (rVV) provide a rapid, easy, and efficient method to introduce Ags into DC, but we observed that rVV infection of DCimm results in blockade of DC maturation in response to all activation signals, including CD40L, monocyte-conditioned medium, LPS, TNF-alpha, and poly(I:C), and failure to induce a CD8(+) response. By contrast, DCmat can be infected with rVV and induce a CD8(+) response, but, having lost phagocytic activity, fail to process the Ag via the exogenous class II pathway. To overcome these limitations, we used the CMV protein pp65 as a model Ag and designed a gene containing the lysosomal-associated membrane protein 1 targeting sequence (Sig-pp65-LAMP1) to target pp65 to the class II compartment. DCmat infected with rVV-Sig-pp65-LAMP1 induced proliferation of pp65-specific CD4(+) clones and efficiently induced a pp65-specific CD4(+) response, suggesting that after DC maturation the intracellular processing machinery for class II remains intact for at least 16 h. Moreover, infection of DCmat with rVV-Sig-pp65-LAMP1 resulted in at least equivalent presentation to CD8(+) cells as infection with rVV-pp65. These results demonstrate that despite rVV interference with DCimm maturation, a single targeting vector can deliver Ags to DCmat for the effective simultaneous stimulation of both CD4(+) and CD8(+) cells.     

Role of the carbonate radical anion in tyrosine nitration and hydroxylation by peroxynitrite

Peroxynitrite has been receiving increasing attention as the pathogenic mediator of nitric oxide cytotoxicity. In most cases, the contribution of peroxynitrite to diseases has been inferred from detection of 3-nitrotyrosine in injured tissues. However, presently it is known that other nitric oxide-derived species can also promote protein nitration. Mechanistic details of protein nitration remain under discussion even in the case of peroxynitrite, although recent literature data strongly suggest a free radical mechanism. Here, we confirm the free radical mechanism of tyrosine modification by peroxynitrite in the presence and in the absence of the bicarbonate-carbon dioxide pair by analyzing the stable tyrosine products and the formation of the tyrosyl radical at pH 5.4 and 7.4. Stable products, 3-nitrotyrosine, 3-hydroxytyrosine, and 3, 3-dityrosine, were identified by high performance liquid chromatography and UV spectroscopy. The tyrosyl radical was detected by continuous-flow and spin-trapping electron paramagnetic resonance (EPR). 3-Hydroxytyrosine was detected at pH 5.4 and its yield decreased in the presence of the bicarbonate-carbon dioxide pair. In contrast, the yields of the tyrosyl radical increased in the presence of the bicarbonate-carbon dioxide pair and correlated with the yields of 3-nitrotyrosine under all tested experimental conditions. Taken together, the results demonstrate that the promoting effects of carbon dioxide on peroxynitrite-mediated tyrosine nitration is due to the selective reactivity of the carbonate radical anion as compared with that of the hydroxyl radical. Colocalization of 3-hydroxytyrosine and 3-nitrotyrosine residues in proteins may be useful to discriminate between peroxynitrite and other nitrating species.     

Plasmodium falciparum soluble extracts potentiate the suppressive function of polyclonal T regulatory cells through activation of TGFβ-mediated signals

Increased numbers of T regulatory cells (Tregs), key mediators of immune homeostasis, were reported in human and murine malaria and it is current opinion that these cells play a role in balancing protective immunity and pathogenesis during infection. However, the mechanisms governing their expansion during malaria infection are not completely defined. In this article we show that soluble extracts of Plasmodium falciparum (PfSEs), but not equivalent preparation of uninfected erythrocytes, induce the differentiation of polyclonally activated CD4(+) cells in Tregs endowed with strong suppressive activity. PfSEs activate latent TGFβ bound on the membrane of Treg cells, thus allowing the cytokine interaction with TGFβ receptor, and inducing Foxp3 gene expression and TGFβ production. The activation of membrane-bound latent TGFβ by PfSEs is significantly reduced by a broad-spectrum metalloproteinases inhibitor with Zn(++) -chelating activity, and completely inhibited by the combined action of such inhibitor and antibodies to a P. falciparum thrombospondin-related adhesive protein (PfTRAP). We conclude that Pf-Zn(++) -dependent proteinases and, to a lesser extent, PfTRAP molecules are involved in the activation of latent TGFβ bound on the membrane of activated Treg cells and suggest that, in malaria infection, this mechanism could contribute to the expansion of Tregs with different antigen specificity.     

Dietary zeolite supplementation reduces oxidative damage and plaque generation in the brain of an Alzheimer's disease mouse model

AimOxidative stress is considered one of the main events that lead to aging and neurodegeneration. Antioxidant treatments used to counteract oxidative damage have been associated with a wide variety of side effects or at the utmost to be ineffective. The aim of the present study was to investigate the antioxidant property of a natural mineral, the tribomechanically micronized zeolite (MZ).Main methodsCell death and oxidative stress were assessed in retinoic acid differentiated SH-SY5Y cells, a neuronal-like cell line, after a pro-oxidant stimulus. In vivo evaluation of antioxidant activity and amyloidogenic processing of beta amyloid have been evaluated in a transgenic model of aging related neurodegeneration, the APPswePS1dE9 transgenic mice (tg mice) after a five-month long period of water supplementation with MZ.Key findingsThe study showed that 24 h of cell pretreatment with MZ (1) protected the cells by radical oxygen species (ROS)-induced cell death and moreover (2) induced a reduction of the mitochondrial ROS production following a pro-oxidant stimulation. Looking for an antioxidant effect of MZ in vivo, we found (3) an increased activity of the endogenous antioxidant enzyme superoxide dismutase (SOD) in the hippocampus of tg mice and (4) a reduction in amyloid levels and plaque load in MZ treated tg mice compared to control tg mice.SignificanceOur results suggest MZ as a novel potential adjuvant in counteracting oxidative stress and plaque accumulation in the field of neurodegenerative diseases.     

The impact of land abandonment on the plant diversity of olive groves

We investigated the effect of woody species’ encroachment on plant diversity changes with regard to vascular plants and bryophytes in traditional olive groves of the Maremma Regional Park (Tuscany, Italy) and assessed cross-taxon correlation between these two taxa. We classified the olive groves into four land use types, representing different successional stages. To describe the evenness of species distribution within a community, we plotted rank-abundance diagrams for each taxon and each land use type. The relationship between the number and cover of vascular plants, therophytes, bryophytes, colonists and phanerophytes in each plot was examined using linear regression. The effects of land use type on vascular plant and bryophyte richness and assemblages were assessed by permutational uni- and multivariate analysis of variance. The congruence in species composition between the two taxa was evaluated using Procrustes analysis. The number of vascular plants, bryophytes and therophytes decreased linearly with increased phanerophyte species cover. The number of species belonging to Thero-Brachypodietea progressively decreased throughout succession. Rank-abundance diagrams and multivariate analysis showed differences between the land use types, which were statistically significant for vascular plants between the traditional olive groves and the other land use types, and for bryophytes between the traditional olive groves and woodlands. PROTEST analysis and NMDS graphs showed a correlation between vascular plant and bryophyte communities. The results suggested that conservation measures are needed in the study area in order to ensure both the maintenance of traditional olive groves of conservation interest and high levels of environmental heterogeneity.     

Forecasting ragweed pollen characteristics with nonparametric regression methods over the most polluted areas in Europe

Nonparametric time-varying regression methods were developed to forecast daily ragweed pollen concentration, and the probability of the exceedance of a given concentration threshold 1 day ahead. Five-day and 10-day predictions of the start and end of the pollen season were also addressed with a nonparametric regression technique combining regression analysis with the method of temperature sum. Our methods were applied to three of the most polluted regions in Europe, namely Lyon (Rhône Valley, France), Legnano (Po River Plain, Italy) and Szeged (Great Plain, Hungary). For a 1-day prediction of both the daily pollen concentration and daily threshold exceedance, the order of these cities from the smallest to largest prediction errors was Legnano, Lyon, Szeged and Legnano, Szeged, Lyon, respectively. The most important predictor for each location was the pollen concentration of previous days. The second main predictor was precipitation for Lyon, and temperature for Legnano and Szeged. Wind speed should be considered for daily concentration at Legnano, and for daily pollen threshold exceedances at Lyon and Szeged. Prediction capabilities compared to the annual cycles for the start and end of the pollen season decreased from west to east. The order of the cities from the lowest to largest errors for the end of the pollen season was Lyon, Legnano, Szeged for both the 5- and 10-day predictions, while for the start of the pollen season the order was Legnano, Lyon, Szeged for 5-day predictions, and Legnano, Szeged, Lyon for 10-day predictions.     

Nitric oxide suppresses tumor cell migration through N-Myc downstream-regulated gene-1 (NDRG1) expression: role of chelatable iron

N-Myc downstream-regulated gene 1 (NDRG1) is a ubiquitous cellular protein that is up-regulated under a multitude of stress and growth-regulatory conditions. Although the exact cellular functions of this protein have not been elucidated, mutations in this gene or aberrant expression of this protein have been linked to both tumor suppressive and oncogenic phenotypes. Previous reports have demonstrated that NDRG1 is strongly up-regulated by chemical iron chelators and hypoxia, yet its regulation by the free radical nitric oxide (^•NO) has never been demonstrated. Herein, we examine the chemical biology that confers NDRG1 responsiveness at the mRNA and protein levels to ^•NO. We demonstrate that the interaction of ^•NO with the chelatable iron pool (CIP) and the appearance of dinitrosyliron complexes (DNIC) are key determinants. Using HCC 1806 triple negative breast cancer cells, we find that NDRG1 is up-regulated by physiological ^•NO concentrations in a dose- and time-dependant manner. Tumor cell migration was suppressed by NDRG1 expression and we excluded the involvement of HIF-1α, sGC, N-Myc, and c-Myc as upstream regulatory targets of ^•NO. Augmenting the chelatable iron pool abolished ^•NO-mediated NDRG1 expression and the associated phenotypic effects. These data, in summary, reveal a link between ^•NO, chelatable iron, and regulation of NDRG1 expression and signaling in tumor cells.     

The tomato terpene synthase gene family

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.