Distinctive role of integrin-mediated adhesion in TNF-induced PKB/Akt and NF-kappaB activation and endothelial cell survival.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine exerting pleiotropic effects on endothelial cells. Depending on the vascular context it can induce endothelial cell activation and survival or death. The microenvironmental cues determining whether endothelial cells will survive or die, however, have remained elusive. Here we report that integrin ligation acts permissive for TNF-induced protein kinase B (PKB/Akt) but not nuclear factor (NF)-kappaB activation. Concomitant activation of PKB/Akt and NF-kappaB is essential for the survival of endothelial cells exposed to TNF. Active PKB/Akt strengthens integrin-dependent endothelial cell adhesion, whereas disruption of actin stress fibers abolishes the protective effect of PKB/Akt. Integrin-mediated adhesion also represses TNF-induced JNK activation, but JNK activity is not required for cell death. The alphaVbeta3/alphaVbeta5 integrin inhibitor EMD121974 sensitizes endothelial cells to TNF-dependent cytotoxicity and active PKB/Akt attenuates this effect. Interferon gamma synergistically enhanced TNF-induced endothelial cell death in all conditions tested. Taken together, these observations reveal a novel permissive role for integrins in TNF-induced PKB/Akt activation and prevention of TNF-induced death distinct of NF-kappaB, and implicate the actin cytoskeleton in PKB/Akt-mediated cell survival. The sensitizing effect of EMD121974 on TNF cytotoxicity may open new perspectives to the therapeutic use of TNF as anticancer agent.

Des antisérums dirigés contre l'antigène carcino-embryonnaire (CEA) peuvent induire une lyse spécifique des cellules de carcinome du côlon par des lymphocytes normaux

Antibody-dependent cell-mediated cytotoxicity (ADCC) against human colon carcinoma cells grown in vitro was demonstrated with two specific rabbit anti-carcinoembryonic antigen (cea) antisera. The same antisera did not lyse the colon carcinoma cells in the presence of complement but without lymphocytes. The normal human lymphocytes in the absence of anti-CEA antiserum had a very low cytotoxic activity during the three hours 51Cr release assay used in this study. Two colon carcinoma cell lines, HT-29 and Co-115, expressing CEA on their surface as demonstrated by immunofluorescence, were significantly lysed in the ADCC test, whereas control tumor cell lines, not expressing CEA, were not affected by the anti-CEA sera and the lymphocytes. The specificity of the reaction was further demonstrated by the inhibition of antibody-dependent cell-mediated cytotoxicity after the addition of increasing amounts of purified CEA to the antiserum. The absorption of the anti-CEA antisera was controlled by radioimmunoassay. Absorption of the antisera by normal lung extracts and red cells of different blood groups did not decrease the cytotoxicity.

NLRC5 deficiency impairs MHC class I-dependent lymphocyte killing by cytotoxic T cells

Purpose/Objective: NLRs are intracellular proteins involved in sensing pathogen- and danger-associated molecular patterns, thereby initiating inflammatory responses or cell death. The function of the family member NLRC5 remains a matter of debate, particularly with respect to NF-jB activation, type I IFN, and MHC class I expression. Materials and methods: To study the function of this NLR in vivo, we generated Nlrc5-deficient mice. Results: We found that NLRC5 deletion led to a mild reduction in MHC class I expression on DCs and an intermediate decrease on B cells, while MHC class I levels were dramatically lowered on T, NKT, and NK cells. Nlrc5-/- lymphocytes showed decreased H-2 gene transcript abundance and, accordingly, NLRC5 was sufficient to drive MHC class I expression in a human lymphoid cell line. Moreover, endogenous NLRC5 localized to the nucleus and occupied the proximal promoter region of H-2 genes. Notably, cytotoxic T cell-mediated elimination of Nlrc5-/- lymphocytes was markedly reduced. In addition, we observed low NLRC5 expression in several murine and human lymphoid-derived tumor cell lines. Conclusions: We found that NLRC5 acts as a key transcriptional regulator of MHC class I genes, in particular in lymphocytes. Loss of NLRC5 expression represents an advantage for evading CD8+ T cellmediated elimination by downmodulation of MHCI levels * a mechanism transformed cells may take advantage of. Therefore, our data support an essential role for NLRs in directing not only innate, but also adaptive immune responses (Staehli F et al. J Immunol 2012).

Pd-catalysed amidation of 2,6-dihalopurine nucleosides. Replacement of iodine at 0 ºC

Pd-catalysed reactions of 2-Cl, 2-Br and 2-I derivatives of a 6-chloropurine nucleoside with benzamide have been compared, using Pd2dba3, Xantphos and Cs2CO3 in toluene, between 20 and 80 °C. The reactivity order was 2-I > 2-Br > 6-Cl ≫ 2-Cl. The 2-I substituent could be replaced even at 0 °C, under conditions disclosed here for the first time. On the other hand, the replacement of the chlorine atom at position 2 (2-Cl) required 110 °C.

Adaptation of anthurium cultivars as cut flowers in a subtropical area

The objective of this work was to evaluate the adaptation of anthurium (Anthurium andraeanum) cultivars as cut flowers in a subtropical area, located in north of Paraná State, Brazil. The Apalai, Ianomami, Kinã, nK 102, Parakanã, Rubi, and Terena cultivars were cultivated in a nursery under 80% shade. The number of leaves and inflorescences, floral stem length, length and width of inflorescences, and spadix length were evaluated. The experimental design was in completely randomized blocks, with six replicates and five plants per plot. During the 18 months evaluated, 'Parakanã' produced 11 inflorescences per plant, whereas the other cultivars produced seven to nine inflorescences per plant. The number of leaves produced per plant was: 'Ianomami', 25; 'Parakanã', 20; 'Apalai' and 'Terena', 15; 'Kinã' and 'Rubi', 11; and 'nK 102', 9. The Apalai, nK 102, Parakanã, and Rubi cultivars are the most recommended as cut flowers; the Ianomami cultivar is not recommended as a cut flower in this region.

Cell-permeable peptides induce dose- and length-dependent cytotoxic effects.

We have explored the threshold of tolerance of three unrelated cell types to treatments with potential cytoprotective peptides bound to Tat(48-57) and Antp(43-58) cell-permeable peptide carriers. Both Tat(48-57) and Antp(43-58) are well known for their good efficacy at crossing membranes of different cell types, their overall low toxicity, and their absence of leakage once internalised. Here, we show that concentrations of up to 100 microM of Tat(48-57) were essentially harmless in all cells tested, whereas Antp(43-58) was significantly more toxic. Moreover, all peptides bound to Tat(48-57) and Antp(43-58) triggered significant and length-dependent cytotoxicity when used at concentrations above 10 microM in all but one cell types (208F rat fibroblasts), irrespective of the sequence of the cargo. Absence of cytotoxicity in 208F fibroblasts correlated with poor intracellular peptide uptake, as monitored by confocal laser scanning fluorescence microscopy. Our data further suggest that the onset of cytotoxicity correlates with the activation of two intracellular stress signalling pathways, namely those involving JNK, and to a lesser extent p38 mitogen-activated protein kinases. These responses are of particular concern for cells that are especially sensitive to the activation of stress kinases. Collectively, these results indicate that in order to avoid unwanted and unspecific cytotoxicity, effector molecules bound to Tat(48-57) should be designed with the shortest possible sequence and the highest possible affinity for their binding partners or targets, so that concentrations below 10 microM can be successfully applied to cells without harm. Considering that cytotoxicity associated to Tat(48-57)- and Antp(43-58) bound peptide conjugates was not restricted to a particular type of cells, our data provide a general framework for the design of cell-penetrating peptides that may apply to broader uses of intracellular peptide and drug delivery.

Proteasome-assisted identification of a SSX-2-derived epitope recognized by tumor-reactive CTL infiltrating metastatic melanoma.

The tumor Ag SSX-2 (HOM-MEL-40) was found by serological identification of Ags by recombinant expression cloning and was shown to be a cancer/testis Ag expressed in a wide variety of tumors. It may therefore represent a source of CD8(+) T cell epitopes useful for specific immunotherapy of cancer. To identify potential SSX-2-derived epitopes that can be recognized by CD8(+) T cells, we used an approach that combined: 1) the in vitro proteasomal digestion of precursor peptides overlapping the complete SSX-2 sequence; 2) the prediction of SSX-2-derived peptides with an appropriate HLA-A2 binding score; and 3) the analysis of a tumor-infiltrated lymph node cell population from an HLA-A2(+) melanoma patient with detectable anti-SSX-2 serum Abs. This strategy allowed us to identify peptide SSX-2(41-49) as an HLA-A2-restricted epitope. SSX2(41-49)-specific CD8(+) T cells were readily detectable in the tumor-infiltrated lymph node population by multimer staining, and CTL clones isolated by multimer-guided cell sorting were able to lyse HLA-A2(+) tumor cells expressing SSX-2.

Circulating Tumor-reactive CD8(+) T cells in melanoma patients contain a CD45RA(+)CCR7(-) effector subset exerting ex vivo tumor-specific cytolytic activity.

To defend the host from malignancies, the immune system can spontaneously raise CD8(+) T-cell responses against tumor antigens. Investigating the functional state of tumor-reactive cytolytic T cells in cancer patients is a key step for understanding the role of these cells in tumor immunosurveillance and for evaluating the potential of immunotherapeutic approaches of vaccination against cancer. In this study we identified a subset of circulating tumor-reactive CD8(+) T lymphocytes, which specifically secreted IFN-gamma after exposition to autologous tumor cell lines in stage IV metastatic melanoma patients. Additional phenotypic characterization using multicolor flow cytometry revealed that a significant fraction of these cells were CD45RA(+)CCR7(-), a phenotype that has been proposed recently to characterize cytolytic effectors potentially able to home into inflamed tissues. In the case of an HLA-A2-expressing patient, the antigen specificity of this population was identified by using HLA-A2/peptide multimers incorporating a tyrosinase-derived peptide. Consistently with their phenotypic characteristics, A2/tyrosinase peptide multimer(+) CD8(+) T cells, isolated by cell sorting, were directly lytic ex vivo and able to specifically recognize tyrosinase-expressing tumor cells. Overall, these results provide the first evidence that a proportion of melanoma patients have circulating tumor-reactive T cells, which are lytic effectors cells.

Local optima networks of hard combinatorial landscapes

Combinatorial optimization involves finding an optimal solution in a finite set of options; many everyday life problems are of this kind. However, the number of options grows exponentially with the size of the problem, such that an exhaustive search for the best solution is practically infeasible beyond a certain problem size. When efficient algorithms are not available, a practical approach to obtain an approximate solution to the problem at hand, is to start with an educated guess and gradually refine it until we have a good-enough solution. Roughly speaking, this is how local search heuristics work. These stochastic algorithms navigate the problem search space by iteratively turning the current solution into new candidate solutions, guiding the search towards better solutions. The search performance, therefore, depends on structural aspects of the search space, which in turn depend on the move operator being used to modify solutions. A common way to characterize the search space of a problem is through the study of its fitness landscape, a mathematical object comprising the space of all possible solutions, their value with respect to the optimization objective, and a relationship of neighborhood defined by the move operator. The landscape metaphor is used to explain the search dynamics as a sort of potential function. The concept is indeed similar to that of potential energy surfaces in physical chemistry. Borrowing ideas from that field, we propose to extend to combinatorial landscapes the notion of the inherent network formed by energy minima in energy landscapes. In our case, energy minima are the local optima of the combinatorial problem, and we explore several definitions for the network edges. At first, we perform an exhaustive sampling of local optima basins of attraction, and define weighted transitions between basins by accounting for all the possible ways of crossing the basins frontier via one random move. Then, we reduce the computational burden by only counting the chances of escaping a given basin via random kick moves that start at the local optimum. Finally, we approximate network edges from the search trajectory of simple search heuristics, mining the frequency and inter-arrival time with which the heuristic visits local optima. Through these methodologies, we build a weighted directed graph that provides a synthetic view of the whole landscape, and that we can characterize using the tools of complex networks science. We argue that the network characterization can advance our understanding of the structural and dynamical properties of hard combinatorial landscapes. We apply our approach to prototypical problems such as the Quadratic Assignment Problem, the NK model of rugged landscapes, and the Permutation Flow-shop Scheduling Problem. We show that some network metrics can differentiate problem classes, correlate with problem non-linearity, and predict problem hardness as measured from the performances of trajectory-based local search heuristics.

Allorestricted T lymphocytes with a high avidity T-cell receptor towards NY-ESO-1 have potent anti-tumor activity.

The cancer-testis antigen NY-ESO-1 has been targeted as a tumor-associated antigen by immunotherapeutical strategies, such as cancer vaccines. The prerequisite for a T-cell-based therapy is the induction of T cells capable of recognizing the NY-ESO-1-expressing tumor cells. In this study, we generated human T lymphocytes directed against the immunodominant NY-ESO-1(157-165) epitope known to be naturally presented with HLA-A*0201. We succeeded to isolate autorestricted and allorestricted T lymphocytes with low, intermediate or high avidity TCRs against the NY-ESO-1 peptide. The avidity of the established CTL populations correlated with their capacity of lysing HLA-A2-positive, NY-ESO-1-expressing tumor cell lines derived from different origins, e.g. melanoma and myeloma. The allorestricted NY-ESO-1-specific T lymphocytes displayed TCRs with the highest avidity and best anti-tumor recognition activity. TCRs derived from allorestricted, NY-ESO-1-specific T cells may be useful reagents for redirecting primary T cells by TCR gene transfer and, therefore, may facilitate the development of adoptive transfer regimens based on TCR-transduced T cells for the treatment of NY-ESO-1-expressing hematological malignancies and solid tumors.

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.

Antigenicity and immunogenicity of Melan-A/MART-1 derived peptides as targets for tumor reactive CTL in human melanoma.

Some cancer patients mount spontaneous T- and B-cell responses against their tumor cells. Autologous tumor reactive CD8 cytolytic T lymphocyte (CTL) and CD4 T-cell clones as well as antibodies from these patients have been used for the identification of genes encoding the target antigens. This knowledge opened the way for new approaches to the immunotherapy of cancer. In this review, we describe the characterization of the structure-function properties of the melanocyte/melanoma tumor antigen Melan-A/MART-1, the assessment of the T-cell repertoire available against this antigen in healthy individuals, and the analysis of naturally acquired and/or vaccine-induced CTL responses to this antigen in patients with metastatic melanoma.

Mechanisms of interaction of therapeutic nanoparticles with cells

Nanoparticles (NPs) have gained a lot of interest in recent years due to their huge potential for applications in industry and medicine. Their unique properties offer a large number of attractive possibilities in the biomedical field, providing innovative tools for diagnosis of diseases and for novel therapies. Nevertheless, a deep understanding of their interactions with living tissues and the knowledge about their possible effects in the human body are necessary for the safe use of nanoparticulate formulations. The aim of this PhD project was to study in detail the interactions of therapeutic NPs with living cells, including cellular uptake and release, cellular localization and transport across the cell layers. Moreover, the effects of NPs on the cellular metabolic processes were determined using adapted in vitro assays. We evaluated the biological effect of several NPs potentially used in the biomedical field, including titanium dioxide (Ti02) NPs, 2-sized fluorescent silica NPs, ultrasmall superparamagnetic iron oxide (USPIO) NPs, either uncoated or coated with oleic acid or with polyvinylamine (aminoPVA) and poly(lactic-co-glycolic acid) - polyethylene-oxide (PLGA-PEO) NPs. We have found that the NPs were internalized by the cells, depending on their size, chemical composition, surface coating and also depending on the cell line considered. The uptake of aminoPVA-coated USPIO NPs by endothelial cells was enhanced in the presence of an external magnetic field. None of the tested USPIO NPs and silica NPs was transported across confluent kidney cell layers or brain endothelial cell layers, even in the presence of a magnetic field. However, in an original endothelium-glioblastoma barrier model which was developed, uncoated USPIO NPs were directly transferred from endothelial cells to glioblastoma cells. Following uptake, Ti02 NPs and uncoated USPIO NPs were released by the kidney cells, but not by the endothelial cells. Furthermore, these NPs induced an oxidative stress and autophagy in brain endothelial cells, possibly associated with their enhanced agglomeration in cell medium. A significant DNA damage was found in brain endothelial cells after their exposure to TiO2NPs. Altogether these results extend the existing knowledge about the effects of NPs on living cells with regard to their physicochemical characteristics and provide interesting tools for further investigation. The development of the in vitro toxicological assays with a special consideration for risk evaluation aims to reduce the use of animal experiments. -Les nanoparticules (NPs) présentent beaucoup d'intérêt dans le domaine biomédical et industriel. Leurs propriétés uniques offrent un grand nombre de possibilités de solutions innovantes pour le diagnostique et la thérapie. Cependant, pour un usage sûr des NPs il est nécessaire d'acquérir une connaissance approfondie des mécanismes d'interactions des NPs avec les tissus vivants et de leur effets sur le corps humain. Le but de ce projet de thèse était d'étudier en détail les mécanismes d'interactions de NPs thérapeutiques avec des cellules vivantes, en particulier les mécanismes d'internalisation cellulaire et leur subséquente sécrétion par les cellules, leur localisation cellulaire, leur transport à travers des couches cellulaires, et l'évaluation des effets de NPs sur le métabolisme cellulaire, en adaptant les méthodes existante d'évaluation cyto-toxico logique s in vitro. Pour ces expériences, les effets biologiques de nanoparticules d'intérêt thérapeutique, telles que des NPs d'oxyde de titane (TiO2), des NPs fluorescents de silicate de 2 tailles différentes, des NPs, d'oxyde de fer super-para-magnétiques ultra-petites (USPIO), soit non- enrobées soit enrobées d'acide oléique ou de polyvinylamine (aminoPVA), et des NPs d'acide poly(lactique-co-glycolique)-polyethylene-oxide (PLGA-PEO) ont été évalués. Les résultats ont démontré que les NPs sont internalisées par les cellules en fonction de leur taille, composition chimique, enrobage de surface, et également du type de cellules utilisées. L'internalisation cellulaire des USPIO NPs a été augmentée en présence d'un aimant externe. Aucune des NPs de fer et de silicate n'a été transportée à travers des couches de cellules épithéliales du rein ou endothéliales du cerveau, même en présence d'un aimant. Cependant, en développant un modèle original de barrière endothélium-glioblastome, un transfert direct de NPs d'oxyde de fer de cellule endothéliale à cellule de glioblastome a été démontré. A la suite de leur internalisation les NPs d'oxyde de fer et de titane sont relâchées par des cellules épithéliales du rein, mais pas des cellules endothéliales du cerveau. Dans les cellules endothéliales du cerveau ces NPs induisent en fonction de leur état d'agglomération un stress oxydatif et des mécanismes d'autophagie, ainsi que des dommages à l'ADN des cellules exposées aux NPs d'oxyde de titane. En conclusion, les résultats obtenus élargissent les connaissances sur les effets exercés par des NPs sur des cellules vivantes et ont permis de développer les outils expérimentaux pour étudier ces effets in vitro, réduisant ainsi le recours à des expériences sur animaux.

Cutting edge: stimulation with the cognate self-antigen induces expression of the Ly49A receptor on self-reactive T cells which modulates their responsiveness.

NK cell self-tolerance is maintained by inhibitory receptors specific for MHC class I molecules. Inhibitory NK receptors are also expressed on memory CD8 T cells but their biological relevance on T cells is unclear. In this study, we describe the expression of the Ly49A receptor on a subset of autoreactive T cells which persist in mice double-transgenic for the lymphocytic choriomeningitis virus-derived peptide gp33 and a TCRalphabeta specific for the gp33. No Ly49A-expressing cells are found in TCRalphabeta single-transgenic mice, indicating that the presence of the autoantigen is required for Ly49A induction. Direct evidence for an Ag-specific initiation of Ly49A expression has been obtained in vitro after stimulation of autoreactive TCRalphabeta T cells with the cognate self-Ag. This expression of Ly49A substantially reduces Ag-specific activation of autoreactive T cells. These findings thus suggest that autoantigen-specific induction of inhibitory NK cell receptors on T cells may contribute to peripheral self-tolerance.

PRDM1 is a tumor suppressor gene in natural killer cell malignancies.

Natural killer cell lymphoma (NKCL) constitutes a rare and aggressive form of non-Hodgkin lymphoma, and there is little insight into its pathogenesis. Here we show that PRDM1 is a tumor suppressor gene in NKCLs that is inactivated by a combination of monoallelic deletion and promoter CpG island hypermethylation. We observed monoallelic deletion of PRDM1 loci in 8 of 18 (44%) NKCL cases. The other allele showed significant promoter methylation in 12 of 17 (71%) cases. In support of its role as a tumor suppressor gene, the reconstitution of PRDM1 in PRDM1-null NK cell lines led to G2/M cell cycle arrest, increased apoptosis, and a strong negative selection pressure with progressive elimination of PRDM1-expressing cells, which was enhanced when IL-2 concentration is limiting. We observed a progressive increase in PRDM1 expression-in particular, PRDM1α-in normal NK cells in response to IL-2 and in normal NK cells activated with an engineered NK cell target, K562-Cl9-mb21, suggesting its role in NK cell homeostasis. In support of this role, knockdown of PRDM1 by shRNA in normal NK cells resulted in the positive selection of these cells. We identified MYC and 4-1BBL as targets of PRDM1 in NK cells. Disruption of homeostatic control by PRDM1 may be an important pathogenetic mechanism for NKCL.