A prospective development study investigating focal irreversible electroporation in men with localised prostate cancer: Nanoknife Electroporation Ablation Trial (NEAT).

INTRODUCTION: Focal therapy may reduce the toxicity of current radical treatments while maintaining the oncological benefit. Irreversible electroporation (IRE) has been proposed to be tissue selective and so might have favourable characteristics compared to the currently used prostate ablative technologies. The aim of this trial is to determine the adverse events, genito-urinary side effects and early histological outcomes of focal IRE in men with localised prostate cancer. METHODS: This is a single centre prospective development (stage 2a) study following the IDEAL recommendations for evaluating new surgical procedures. Twenty men who have MRI-visible disease localised in the anterior part of the prostate will be recruited. The sample size permits a precision estimate around key functional outcomes. Inclusion criteria include PSA ≤ 15 ng/ml, Gleason score ≤ 4 + 3, stage T2N0M0 and absence of clinically significant disease outside the treatment area. Treatment delivery will be changed in an adaptive iterative manner so as to allow optimisation of the IRE protocol. After focal IRE, men will be followed during 12 months using validated patient reported outcome measures (IPSS, IIEF-15, UCLA-EPIC, EQ-5D, FACT-P, MAX-PC). Early disease control will be evaluated by mpMRI and targeted transperineal biopsy of the treated area at 6 months. DISCUSSION: The NEAT trial will assess the early functional and disease control outcome of focal IRE using an adaptive design. Our protocol can provide guidance for designing an adaptive trial to assess new surgical technologies in the challenging landscape of health technology assessment in prostate cancer treatment.

Characterising the role of T-cell subsets in experimental allograft rejection and transplantation tolerance

SUMMARY : The recognition by recipient T cells of the allograft major histocompatibility complex (MHC)mismatched antigens is the primary event that ultimately leads to rejection. In the transplantation setting, circulating alloreactive CD4+ T cells play a central role in the initiation and the coordination of the immune response and can initiate the rejection of an allograft via three distinct pathways: the direct, indirect and the recently described semi-direct pathway. However, the exact role of individual CD4+ T-cell subsets in the development of allograft rejection is not clearly defined. Furthermore, besides pathogenic effector T cells, a new subset of T cells with regulatory properties, the CD4+CD25+Foxp3+ (Treg) cells, has come under increased scrutiny over the last decade. The experiments presented in this thesis were designed to better define the phenotype and functional characteristics of CD4+ T-cell subsets and Treg cells in vitro and in vivo in a marine adoptive transfer and skin transplantation model. As Treg cells play a key role in the induction and maintenance of peripheral transplantation tolerance, we have explored whether donor-antigen specific Treg cells could be expanded in vitro. Here we describe a robust protocol for the ex-vivo generation and expansion of antigen-specific Treg cells, without loss of their characteristic phenotype and suppressive function. In our in vivo transplantation model, antigen-specific Treg cells induced donor-specific tolerance to skin allografts in lymphopenic recipients and significantly delayed skin graft rejection in wild-type mice in the absence of any other immunosuppression. Naïve and memory CD4+ T cells have distinct phenotypes, effector functions and in vivo homeostatsis, and thus may play different roles in anti-donor immunity after transplantation. We have analyzed in vitro and in vivo primary alloresponses of naïve and cross-reactive memory CD4+ T cells. We found that the CD4+CD45RBlo memory T-cell pool was heterogeneous and contained cells with regulatory potentials, both in the CD4+CD25+ and CD4+CD25- populations. CD4+ T cells capable of inducing strong primary alloreactive responses in vitro and rejection of a first allograft in vivo were mainly contained within the CD45RBhi naïve CD4+ T-cell compartment. Taken together, the work described in this thesis provides new insights into the mechanisms that drive allograft rejection or donor-specific transplantation tolerance. These results will help to optimise current clinical immunosuppressive regimens used after solid organ transplantation and design new immunotherapeutic strategies to prevent transplant rejection. RÉSUMÉ : ROLE DES SOUS-POPULATIONS DE CELLULES T DANS LE REJET DE GREFFE ET L'INDUCTION DE TOLERANCE EN TRANSPLANTATION La reconnaissance par les cellules T du receveur des alloantigènes du complexe majeur d'histocompatibilité (CMIT) présentés par une greffe allogénique, est le premier événement qui aboutira au rejet de l'organe greffé. Dans le contexte d'une transplantation, les cellules alloréactives T CD4+ circulantes jouent un rôle central dans l'initiation et la coordination de 1a réponse immune, et peuvent initier le rejet par 3 voies distinctes : la voie directe, indirecte et la voie servi-directe, plus récemment décrite. Toutefois, le rôle exact des sous-populations de cellules T CD4+ dans les différentes étapes menant au rejet d'une allogreffe n'est pas clairement établi. Par ailleurs, hormis les cellules T effectrices pathogéniques, une sous-population de cellules T ayant des propriétés régulatrices, les cellules T CD4+CD25+Foxp3+ (Treg), a été nouvellement décrite et est intensément étudiée depuis environ dix ans. Les expériences présentées dans cette thèse ont été planifiées afin de mieux définir le phénotype et les caractéristiques fonctionnels des sous-populations de cellules T CD4+ et des Treg in vitro et in vivo dans un modèle marin de transfert adoptif de cellules et de transplantation de peau. Comme les cellules Treg jouent un rôle clé dans l'induction et le maintien de la tolérance périphérique en transplantation, nous avons investigué la possibilité de multiplier in vitro des cellules Treg avec spécificité antigénique pour le donneur. Nous décrivons ici un protocole reproductible pour la génération et l'expansion ex-vivo de cellules Treg avec spécificité antigénique, sans perte de leur phénotype caractéristique et de leur fonction suppressive. Dans notre modèle in vivo de transplantation de peau, ces cellules Treg pouvaient induire une tolérance spécifique vis-à-vis du donneur chez des souris lymphopéniques, et, chez des souris normales non-lymphopéniques ces Treg ont permis de retarder significativement le rejet en l'absence de tout traitement immunosuppresseur. Les cellules T CD4+ naïves et mémoires se distinguent par leur phénotype, fonction effectrice et leur homéostasie in vivo, et peuvent donc moduler différemment la réponse immune contre le donneur après transplantation. Nous avons analysé in vitro et in vivo les réponses allogéniques primaires de cellules T CD4+ naïves et mémoires non-spécifiques (cross-réactives). Nos résultats ont montré que le pool de cellules T CD4+CD45RB'° mémoires était hétérogène et contenait des cellules avec un potentiel régulateur, aussi bien parmi la sous-population de cellules CD4+CD25+ que CD4+CD25+. Les cellules T CD4+ capables d'induire une alloréponse primaire intense in vitro et le rejet d'une première allogreffe in vivo étaient essentiellement contenues dans le pool de cellules T CD4+CD45RBhi naïves. En conclusion, le travail décrit dans cette thèse amène un nouvel éclairage sur les mécanismes responsables du rejet d'une allogreffe ou de l'induction de tolérance en transplantation. Ces résultats permettront d'optimaliser les traitements immunosuppresseurs utilisés en transplantation clinique et de concevoir des nouvelles stratégies irnmuno-thérapeutiques pour prévenir le rejet de greffe allogénique.

Convenient synthesis of C75, an inhibitor of FAS and CPT1

C75 is a synthetic racemic α-methylene-γ-butyrolactone exhibiting anti-tumoral properties in vitro and in vivo as well as inducing hypophagia and weight loss in rodents. These interesting properties are thought to be a consequence of the inhibition of the key enzymes FAS and CPT1 involved in lipid metabolism. The need for larger amounts of this compound for biological evaluation prompted us to develop a convenient and reliable route to multigram quantities of C75 from easily available ethyl penta-3,4-dienoate 6. A recently described protocol for the addition of 6 to a mixture of dicyclohexylborane and nonanal followed by acidic treatment of the crude afforded lactone 8, as a mixture of cis and trans isomers, in good yield. The DBU-catalyzed isomerization of the methyl esters 9 arising from 8 gave a 10:1 trans/cis mixture from which the trans isomer was isolated and easily transformed into C75. The temporary transformation of C75 into a phenylseleno ether derivative makes its purification, manipulation and storage easier.

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

Besides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis.

Connexins: key mediators of endocrine function.

The appearance of multicellular organisms imposed the development of several mechanisms for cell-to-cell communication, whereby different types of cells coordinate their function. Some of these mechanisms depend on the intercellular diffusion of signal molecules in the extracellular spaces, whereas others require cell-to-cell contact. Among the latter mechanisms, those provided by the proteins of the connexin family are widespread in most tissues. Connexin signaling is achieved via direct exchanges of cytosolic molecules between adjacent cells at gap junctions, for cell-to-cell coupling, and possibly also involves the formation of membrane "hemi-channels," for the extracellular release of cytosolic signals, direct interactions between connexins and other cell proteins, and coordinated influence on the expression of multiple genes. Connexin signaling appears to be an obligatory attribute of all multicellular exocrine and endocrine glands. Specifically, the experimental evidence we review here points to a direct participation of the Cx36 isoform in the function of the insulin-producing β-cells of the endocrine pancreas, and of the Cx40 isoform in the function of the renin-producing juxtaglomerular epithelioid cells of the kidney cortex.

Detecting, quantifying and adjusting for publication bias in meta-analyses: protocol of a systematic review on methods.

BACKGROUND: Health professionals and policymakers aspire to make healthcare decisions based on the entire relevant research evidence. This, however, can rarely be achieved because a considerable amount of research findings are not published, especially in case of 'negative' results - a phenomenon widely recognized as publication bias. Different methods of detecting, quantifying and adjusting for publication bias in meta-analyses have been described in the literature, such as graphical approaches and formal statistical tests to detect publication bias, and statistical approaches to modify effect sizes to adjust a pooled estimate when the presence of publication bias is suspected. An up-to-date systematic review of the existing methods is lacking. METHODS/DESIGN: The objectives of this systematic review are as follows:âeuro¢ To systematically review methodological articles which focus on non-publication of studies and to describe methods of detecting and/or quantifying and/or adjusting for publication bias in meta-analyses.âeuro¢ To appraise strengths and weaknesses of methods, the resources they require, and the conditions under which the method could be used, based on findings of included studies.We will systematically search Web of Science, Medline, and the Cochrane Library for methodological articles that describe at least one method of detecting and/or quantifying and/or adjusting for publication bias in meta-analyses. A dedicated data extraction form is developed and pilot-tested. Working in teams of two, we will independently extract relevant information from each eligible article. As this will be a qualitative systematic review, data reporting will involve a descriptive summary. DISCUSSION: Results are expected to be publicly available in mid 2013. This systematic review together with the results of other systematic reviews of the OPEN project (To Overcome Failure to Publish Negative Findings) will serve as a basis for the development of future policies and guidelines regarding the assessment and handling of publication bias in meta-analyses.

The short-rooted phenotype of the brevis radix mutant partly reflects root abscisic acid hypersensitivity.

To gain further insight into abscisic acid (ABA) signaling and its role in growth regulation, we have screened for Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to ABA-mediated root growth inhibition. As a result, we have identified a loss-of-function allele of BREVIS RADIX (BRX) in the Columbia background, named brx-2, which shows enhanced response to ABA-mediated inhibition of root growth. BRX encodes a key regulator of cell proliferation and elongation in the root, which has been implicated in the brassinosteroid (BR) pathway as well as in the regulation of auxin-responsive gene expression. Mutants affected in BR signaling that are not impaired in root growth, such as bes1-D, bzr1-D, and bsu1-D, also showed enhanced sensitivity to ABA-mediated inhibition of root growth. Triple loss-of-function mutants affected in PP2Cs, which act as negative regulators of ABA signaling, showed impaired root growth in the absence of exogenous ABA, indicating that disturbed regulation of ABA sensitivity impairs root growth. In agreement with this result, diminishing ABA sensitivity of brx-2 by crossing it with a 35S:HAB1 ABA-insensitive line allowed significantly higher recovery of root growth after brassinolide treatment. Finally, transcriptomic analysis revealed that ABA treatment negatively affects auxin signaling in wild-type and brx-2 roots and that ABA response is globally altered in brx-2. Taken together, our results reveal an interaction between BRs, auxin, and ABA in the control of root growth and indicate that altered sensitivity to ABA is partly responsible for the brx short-root phenotype.

Improved flooding of broadcast messages using extended multipoint relaying

A common operation in wireless ad hoc networks is the flooding of broadcast messages to establish network topologies and routing tables. The flooding of broadcast messages is, however, a resource consuming process. It might require the retransmission of messages by most network nodes. It is, therefore, very important to optimize this operation. In this paper, we first analyze the multipoint relaying (MPR) flooding mechanism used by the Optimized Link State Routing (OLSR) protocol to distribute topology control (TC) messages among all the system nodes. We then propose a new flooding method, based on the fusion of two key concepts: distance-enabled multipoint relaying and connected dominating set (CDS) flooding. We present experimental simulationsthat show that our approach improves the performance of previous existing proposals.

Role of CD4+CD25+ regulatory T cells in the antigen specific induction of tolerance "via" the nasal route

ABSTRACT Asthma is a complex inflammatory syndrome caused by environmental factors in predisposed individuals (atopics). Its severity correlates with the presence of activated T lymphocytes and eosinophils in the bronchoalveolar lavage fluid (BALF). Induction of tolerance via the nasal route results in reduced recruitment of eosinophils into BALF upon challenge, inhibition of TH2 pro-inflammatory cytokine secretion and T cell hyporesponsiveness. Recently, CD4+CD25+ natural regulatory T cells (Treg) were proposed as key players in controlling the development of asthma and allergic disease. The objective of the present study is to investigate the role of CD4+CD25+ regulatory T cells in the mechanisms leading to tolerance in an established model of asthma. In this goal we depleted CD4+CD25+ T cells at different times during asthma and tolerance induction protocol in mice and looked at efficiency of tolerization (intranasal application of high dose of allergen) in the absence of natural Tregs. First, ovalbumin-sensitized mice were depleted of CD25+ T cells by intraperitoneal injection of anti-CD25 mAb (PC61) either for along-term (repeated injections of anti-CD25 from day 31 until the end of the protocol) or a short-term period (single injection of anti-CD25 before or after tolerance induction). We demonstrated that the long-term depletion of CD4+CD25+ T cells severely hampered tolerance induction (marked enhancement in eosinophil recruitment into BALF and a vigorous antigen specific T cell response to OVA upon allergen challenge) whereas transient depletions were not sufficient to do so. We then characterized T cell subsets by flow cytometry and observed that a large part of CD4+CD25+ T cells express Foxp3, an established marker of regulatory T cells. We also tested in-vitro suppressor activity of CD4+CD25+ T cells from tolerized mice by cell proliferation assay in coculture and observed a strong suppressive activity. Our data suggest that CD4+CD25+ T cells with regulatory properties play a crucial role in the induction of tolerance via the nasal route. The relationship between CD25+ natural Treg and inducible IL-10+ TRl-type Treg will have to be defined. RESUME L'asthme est un syndrome inflammatoire complexe provoqué par des facteurs environnementaux chez des individus génétiquement prédisposés (atopiques). Sa sévérité corrèle avec la présence des lymphocytes T activés et d'éosinophiles dans le lavage bronchoalvéolaire (BAL). L'induction de la tolérance par la voie nasale résulte en une diminution du recrutement des eosinophils dans le BAL, une inhibition de la sécrétion de cytokines pro-inflammatoires de type TH2 et de l'hypo-réponse des cellules T à l'allergène. Récemment, les cellules régulatrices «naturelles » de type CD4+CD25+ T (Tregs) ont été proposées comme acteurs essentiels dans le développement de l'asthme et de l'allergie. L'objectif de cette étude est d'étudier le rôle des cellules régulatrices CD4+CD25+ T dans les mécanismes menant à la tolérance dans un modèle établi d'asthme. Dans ce but nous avons déplété les cellules de CD4+CD25+ T à différents temps au cours du protocole d'induction d'asthme et de tolérance et nous avons regardé l'efficacité de l'induction de tolérance (application intranasale d'une dose importante d'allergène) en l'absence de Tregs. Dans un premier temps des souris sensibilisées à l'ovalbumine (OVA) ont été déplétées en cellules CD25+ T par l'injection intrapéritonéale d'anti-CD25 mAb (PC61) pour une longue période (injections répétées d'anti-CD25 du jour 31 jusqu'à la fin du protocole) ou pour une courte période (injection unique d'anti-CD25 avant ou après l'induction de tolérance). Nous avons démontré que la déplétion à long t erme des cellules de CD4+CD25+ T a empêché l'induction de tolérance (recrutement accru d'éosinophiles dans le BAL et une réponse vigoureuse des cellules T spécifiques de l'antigène après exposition à l'allergène) tandis des déplétions à court-terme n'ont pas cet effet. Nous avons ensuite caractérisé des sous-populations de cellules T par cytométrie de flux. Nous avons observé que la majorité des cellules CD4+CD25+ T expriment Foxp3, un marqueur établi des cellules régulatrices. Nous avons également examiné in vitro l'activité régulatrice des cellules T CD4+CD25+ issues de souris tolérisées. La prolifération de cellules T en coculture a démontré une forte activité suppressive des cellules CD4+CD25+. Nos données suggèrent que des cellules T CD4+CD25+ ayant des propriétés régulatrices jouent un rôle crucial dans l'induction de la tolérance par la voie nasale. Le rapport entre les cellules régulatrices naturelles CD4+CD25+ et les cellules régulatrices inductible de type TR1 I1-10+ devra être défini. RESUME DESTINE A UN LARGE PUBLIC L'asthme est une maladie inflammatoire des bronches, caractérisée par des crises de dyspnée (gêne respiratoire) témoignant d'une activation brutale des muscles bronchoconstricteurs, auxquelles s'associent un oedème et une hypersécrétion des muqueuses des voies aériennes ainsi qu'une importante production d'anticorps de l'allergie (IgE). Chez la plupart des enfants atteints et chez près de la moitié des adultes concernés par l'asthme, c'est une allergie à des substances présentes dans l'air environnant (acariens, pollens ou poils d'animaux) qui est à l'origine de la maladie. . Le traitement actuel de l'asthme repose d'une part sur le soulagement des symptômes grâce à des produits à base de stéroïdes ou des bronchodilatateurs. D'autre part, l'immunothérapie spécifique (aussi appelée désensibilisation) permet d'améliorer l'asthme et de «reprogrammer» le système immunitaire. C'est à ce jour, le seul moyen connu de faire régresser une allergie. Cependant l'immunothérapie prend beaucoup de temps (3 à 5 ans) et ne marche pas à tous les coups ni pour tous les antigènes. Il est donc important de mieux comprendre les mécanismes impliqués lors d'un tel traitement afin d'en améliorer l'efficacité. Af n de pouvoir investiguer en détail ces mécanismes des modèles d'immunothérapie ont été mis au point chez la souris. Notre étude se base sur un modèle d'asthme allergique chez la souris. Des souris sont rendues allergiques à l'ovalbumine (OVA) et présentent alors les caractéristiques majeures de l'asthme humain (recrutement de cellules inflammatoires dans les poumons, augmentation de la production d'IgE et de la résistance des bronches aux flux respiratoires). Ces souris asthmatiques une fois traitées par l'application nasale d'OVA (forme d'immunothérapie muqueuse) ne développent plus de réaction allergique lors d'une ré-exposition à l'allergène. Notre hypothèse est que cette «guérison» (tolérance) est liée à l'action de cellules (lymphocytes T CD4) dites «régulatrices» et caractérisées par le marqueur CD25. Pour le démontrer, nous avons éliminé ces cellules «régulatrices» CD25 de nos souris asthmatiques grâce à un anticorps monoclonal spécifique. Nous n'avons dès lors plus été en mesure d'induire une tolérance à l'allergène. Ceci suggère donc un rôle clé des cellules «régulatrices» T CD4+CD25+ dans la réussite de l'immunothérapie nasale dans notre modèle. Nos résultats n'excluent pas la participation d'autres cellules telles que les lymphocytes producteurs d'IL-10 (lymphocytes régulateurs induits). Le rôle respectif de ces sous-populations régulatrices devra être examiné dans les études à venir. Une meilleure maîtrise des mécanismes de régulation pourrait s'avérer cruciale pour améliorer les thérapies de l'asthme.

Anonymous Routing Protocol in decentralized networks

Peer-reviewed

Visualizing key authenticity: turning your face into your public key

La informació biomètrica s'ha convertit en una tecnologia complementària a la criptografia que permet administrar còmodament les dades criptogràfiques. Són útils dues necessitats importants: en primer lloc, posar aquestes dades sempre a mà i, a més, fent fàcilment identificable el seu legítim propietari. En aquest article es proposa un sistema que integra la signatura biomètrica de reconeixement facial amb un esquema de signatura basat en la identitat, de manera que la cara de l'usuari esdevé la seva clau pública i la ID del sistema. D'aquesta manera, altres usuaris poden verificar els missatges utilitzant fotos del remitent, proporcionant un intercanvi raonable entre la seguretat del sistema i la usabilitat, així com una manera molt més senzilla d'autenticar claus públiques i processos de distribució.

A Secure Cooperative Sensing Protocol for Cognitive Radio Networks

Cognitive radio networks sense spectrum occupancyand manage themselves to operate in unused bands without disturbing licensed users. Spectrum sensing is more accurate if jointly performed by several reliable nodes. Even though cooperative sensing is an active area of research, the secureauthentication of local sensing reports remains unsolved, thus empowering false results. This paper presents a distributed protocol based on digital signatures and hash functions, and ananalysis of its security features. The system allows determining a final sensing decision from multiple sources in a quick and secure way.

Secure Dynamic MANET On-demand (SEDYMO) Routing Protocol

This paper describes the state of the art of secure ad hoc routing protocols and presents SEDYMO, a mechanism to secure a dynamic multihop ad hoc routing protocol. The proposed solution defeats internal and external attacks usinga trustworthiness model based on a distributed certification authority. Digital signatures and hash chains are used to ensure the correctness of the protocol. The protocol is compared with other alternatives in terms of security strength, energy efficiency and time delay. Both computational and transmission costs are considered and it is shown that the secure protocol overhead is not a critical factor compared to the high network interface cost.