Highly diverse T cell recognition of a single Plasmodium berghei peptide presented by a series of mutant H-2Kd molecules.

We have tested 21 independent CTL clones for recognition of a single peptide derived from the Plasmodium berghei circumsporozoite protein in the context of 13 mutants of the murine MHC class I molecule H-2Kd. In this series of Kd mutants, amino acid residues located on the upper surface of the alpha-helices were individually substituted by alanine. Remarkably, most clones displayed individual recognition patterns on the Kd mutants. We had previously found that this series of CTL clones was likewise highly diverse in terms of both TCR primary structure and peptide fine specificity. Our data thus reinforce the concept that multiple T cell epitopes are available on the surface of a single peptide-MHC class I complex for recognition by specific TCR.

Chemodiversity and molecular plasticity: recognition processes as explored by property spaces.

In the last few years, a need to account for molecular flexibility in drug-design methodologies has emerged, even if the dynamic behavior of molecular properties is seldom made explicit. For a flexible molecule, it is indeed possible to compute different values for a given conformation-dependent property and the ensemble of such values defines a property space that can be used to describe its molecular variability; a most representative case is the lipophilicity space. In this review, a number of applications of lipophilicity space and other property spaces are presented, showing that this concept can be fruitfully exploited: to investigate the constraints exerted by media of different levels of structural organization, to examine processes of molecular recognition and binding at an atomic level, to derive informative descriptors to be included in quantitative structure--activity relationships and to analyze protein simulations extracting the relevant information. Much molecular information is neglected in the descriptors used by medicinal chemists, while the concept of property space can fill this gap by accounting for the often-disregarded dynamic behavior of both small ligands and biomacromolecules. Property space also introduces some innovative concepts such as molecular sensitivity and plasticity, which appear best suited to explore the ability of a molecule to adapt itself to the environment variously modulating its property and conformational profiles. Globally, such concepts can enhance our understanding of biological phenomena providing fruitful descriptors in drug-design and pharmaceutical sciences.

Functional analysis of HLA-A*0201/Melan-A peptide multimer+ CD8+ T cells isolated from an HLA-A*0201- donor: exploring tumor antigen allorestricted recognition.

Recent studies in mouse models have suggested that genetic transfer of tumor antigen-specific high affinity T cell receptors (TCR) into host lymphocytes could be a viable strategy for the rapid induction of tumor-specific immunity. A previously proposed approach for the isolation of such TCRs consists in circumventing tolerance to self-restricting HLA/peptide complexes by deriving them from PMBCs of allogenic donors. Towards this aim, we used fluorescent HLA-A2 class-I/peptide soluble multimers to isolate A2-restricted CD8+ T cells specific for a previously described Melan-A peptide enhanced analog (Melan-A 26-35 A27L) from an HLA-A*0201 (A2) negative donor. We isolated two distinct groups of Melan-A 26-35 A27L-specific clones. Clones from the first group recognized the analog peptide with high avidity but showed very low recognition of Melan-A parental peptides. In contrast, clones from the second group efficiently recognized Melan-A parental peptides. Surprisingly however, most clones recognized not only A2+ Melan-A+ targets, but also A2+ Melan-A- targets suggesting that they can also recognize endogenous peptides other than Melan-A. In addition, one clone showed full cross-recognition of an antigenically unrelated peptide. Together, our data show that HLA-A2/peptide multimers can be successfully used for the isolation of allorestricted CD8+ T cells reactive with tumor antigen-derived peptides. However, as the cross-reactivity of these apparently peptide-specific allorestricted TCRs is presently unpredictable, a careful in vitro analysis of their reactivity to the host's normal cells is recommended.

The evolution of unicoloniality in ant societies : recognition, dispersal and population genetic structure

Introduction Societies of ants, bees, wasps and termites dominate many terrestrial ecosystems (Wilson 1971). Their evolutionary and ecological success is based upon the regulation of internal conflicts (e.g. Ratnieks et al. 2006), control of diseases (e.g. Schmid-Hempel 1998) and individual skills and collective intelligence in resource acquisition, nest building and defence (e.g. Camazine 2001). Individuals in social species can pass on their genes not only directly trough their own offspring, but also indirectly by favouring the reproduction of relatives. The inclusive fitness theory of Hamilton (1963; 1964) provides a powerful explanation for the evolution of reproductive altruism and cooperation in groups with related individuals. The same theory also led to the realization that insect societies are subject to internal conflicts over reproduction. Relatedness of less-than-one is not sufficient to eliminate all incentive for individual selfishness. This would indeed require a relatedness of one, as found among cells of an organism (Hardin 1968; Keller 1999). The challenge for evolutionary biology is to understand how groups can prevent or reduce the selfish exploitation of resources by group members, and how societies with low relatedness are maintained. In social insects the evolutionary shift from single- to multiple queens colonies modified the relatedness structure, the dispersal, and the mode of colony founding (e.g. (Crozier & Pamilo 1996). In ants, the most common, and presumably ancestral mode of reproduction is the emission of winged males and females, which found a new colony independently after mating and dispersal flights (Hölldobler & Wilson 1990). The alternative reproductive tactic for ant queens in multiple-queen colonies (polygyne) is to seek to be re-accepted in their natal colonies, where they may remain as additional reproductives or subsequently disperse on foot with part of the colony (budding) (Bourke & Franks 1995; Crozier & Pamilo 1996; Hölldobler & Wilson 1990). Such ant colonies can contain up to several hundred reproductive queens with an even more numerous workforce (Cherix 1980; Cherix 1983). As a consequence in polygynous ants the relatedness among nestmates is very low, and workers raise brood of queens to which they are only distantly related (Crozier & Pamilo 1996; Queller & Strassmann 1998). Therefore workers could increase their inclusive fitness by preferentially caring for their closest relatives and discriminate against less related or foreign individuals (Keller 1997; Queller & Strassmann 2002; Tarpy et al. 2004). However, the bulk of the evidence suggests that social insects do not behave nepotistically, probably because of the costs entailed by decreased colony efficiency or discrimination errors (Keller 1997). Recently, the consensus that nepotistic behaviour does not occur in insect colonies was challenged by a study in the ant Formica fusca (Hannonen & Sundström 2003b) showing that the reproductive share of queens more closely related to workers increases during brood development. However, this pattern can be explained either by nepotism with workers preferentially rearing the brood of more closely related queens or intrinsic differences in the viability of eggs laid by queens. In the first chapter, we designed an experiment to disentangle nepotism and differences in brood viability. We tested if workers prefer to rear their kin when given the choice between highly related and unrelated brood in the ant F. exsecta. We also looked for differences in egg viability among queens and simulated if such differences in egg viability may mistakenly lead to the conclusion that workers behave nepotistically. The acceptance of queens in polygnous ants raises the question whether the varying degree of relatedness affects their share in reproduction. In such colonies workers should favour nestmate queens over foreign queens. Numerous studies have investigated reproductive skew and partitioning of reproduction among queens (Bourke et al. 1997; Fournier et al. 2004; Fournier & Keller 2001; Hammond et al. 2006; Hannonen & Sundström 2003a; Heinze et al. 2001; Kümmerli & Keller 2007; Langer et al. 2004; Pamilo & Seppä 1994; Ross 1988; Ross 1993; Rüppell et al. 2002), yet almost no information is available on whether differences among queens in their relatedness to other colony members affects their share in reproduction. Such data are necessary to compare the relative reproductive success of dispersing and non-dispersing individuals. Moreover, information on whether there is a difference in reproductive success between resident and dispersing queens is also important for our understanding of the genetic structure of ant colonies and the dynamics of within group conflicts. In chapter two, we created single-queen colonies and then introduced a foreign queens originating from another colony kept under similar conditions in order to estimate the rate of queen acceptance into foreign established colonies, and to quantify the reproductive share of resident and introduced queens. An increasing number of studies have investigated the discrimination ability between ant workers (e.g. Holzer et al. 2006; Pedersen et al. 2006), but few have addressed the recognition and discrimination behaviour of workers towards reproductive individuals entering colonies (Bennett 1988; Brown et al. 2003; Evans 1996; Fortelius et al. 1993; Kikuchi et al. 2007; Rosengren & Pamilo 1986; Stuart et al. 1993; Sundström 1997; Vásquez & Silverman in press). These studies are important, because accepting new queens will generally have a large impact on colony kin structure and inclusive fitness of workers (Heinze & Keller 2000). In chapter three, we examined whether resident workers reject young foreign queens that enter into their nest. We introduced mated queens into their natal nest, a foreign-female producing nest, or a foreign male-producing nest and measured their survival. In addition, we also introduced young virgin and mated queens into their natal nest to examine whether the mating status of the queens influences their survival and acceptance by workers. On top of polgyny, some ant species have evolved an extraordinary social organization called 'unicoloniality' (Hölldobler & Wilson 1977; Pedersen et al. 2006). In unicolonial ants, intercolony borders are absent and workers and queens mix among the physically separated nests, such that nests form one large supercolony. Super-colonies can become very large, so that direct cooperative interactions are impossible between individuals of distant nests. Unicoloniality is an evolutionary paradox and a potential problem for kin selection theory because the mixing of queens and workers between nests leads to extremely low relatedness among nestmates (Bourke & Franks 1995; Crozier & Pamilo 1996; Keller 1995). A better understanding of the evolution and maintenance of unicoloniality requests detailed information on the discrimination behavior, dispersal, population structure, and the scale of competition. Cryptic genetic population structure may provide important information on the relevant scale to be considered when measuring relatedness and the role of kin selection. Theoretical studies have shown that relatedness should be measured at the level of the `economic neighborhood', which is the scale at which intraspecific competition generally takes place (Griffin & West 2002; Kelly 1994; Queller 1994; Taylor 1992). In chapter four, we conducted alarge-scale study to determine whether the unicolonial ant Formica paralugubris forms populations that are organised in discrete supercolonies or whether there is a continuous gradation in the level of aggression that may correlate with genetic isolation by distance and/or spatial distance between nests. In chapter five, we investigated the fine-scale population structure in three populations of F. paralugubris. We have developed mitochondria) markers, which together with the nuclear markers allowed us to detect cryptic genetic clusters of nests, to obtain more precise information on the genetic differentiation within populations, and to separate male and female gene flow. These new data provide important information on the scale to be considered when measuring relatedness in native unicolonial populations.

Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the cross-recognition.

Cytolytic T lymphocyte recognition of the murine cytomegalovirus nonstructural immediate-early protein pp89 expressed by recombinant vaccinia virus.

The murine immediate-early (IE) protein pp89 is a nonstructural virus-encoded phosphoprotein residing in the nucleus of infected cells, where it acts as transcriptional activator. Frequency analysis has shown that in BALB/c mice the majority of virus-specific CTL recognize IE antigens. The present study was performed to assess whether pp89 causes membrane antigen expression detected by IE-specific CTL. Site-directed mutagenesis has been used to delete the introns from gene ieI, encoding pp89, for subsequent integration of the continuous coding sequence into the vaccinia virus genome. After infection with the vaccinia recombinant, the authentic pp89 was expressed in cells that became susceptible to lysis by an IE-specific CTL clone. Priming of mice with the vaccinia recombinant sensitized polyclonal CTL that recognized MCMV-infected cells and transfected cells expressing pp89. Thus, a herpesviral IE polypeptide with essential function in viral transcriptional regulation can also serve as a dominant antigen for the specific CTL response of the host.

Recognition of major histoincompatibilities after transplantation with marrow from HLA closely matched donors.

BACKGROUND: To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. METHODS: Patient 1 was mismatched for one "ABDR-antigen" (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an "HLA-A subtype" (A*0213 versus A*0201), whereas patient 3 was mismatched for HLA-C (HLA-C*0501 versus HLA-C*0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. RESULTS: Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. CONCLUSIONS: Independent of whether the incompatibility consisted of a "full" mismatch, a "subtype" mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.

Low TCR avidity and lack of tumor cell recognition in CD8(+) T cells primed with the CEA-analogue CAP1-6D peptide.

The use of "altered peptide ligands" (APL), epitopes designed for exerting increased immunogenicity as compared with native determinants, represents nowadays one of the most utilized strategies for overcoming immune tolerance to self-antigens and boosting anti-tumor T cell-mediated immune responses. However, the actual ability of APL-primed T cells to cross-recognize natural epitopes expressed by tumor cells remains a crucial concern. In the present study, we show that CAP1-6D, a superagonist analogue of a carcinoembriyonic antigen (CEA)-derived HLA-A*0201-restricted epitope widely used in clinical setting, reproducibly promotes the generation of low-affinity CD8(+) T cells lacking the ability to recognized CEA-expressing colorectal carcinoma (CRC) cells. Short-term T cell cultures, obtained by priming peripheral blood mononuclear cells from HLA-A*0201(+) healthy donors or CRC patients with CAP1-6D, were indeed found to heterogeneously cross-react with saturating concentrations of the native peptide CAP1, but to fail constantly lysing or recognizing through IFN- gamma release CEA(+)CRC cells. Characterization of anti-CAP1-6D T cell avidity, gained through peptide titration, CD8-dependency assay, and staining with mutated tetramers (D227K/T228A), revealed that anti-CAP1-6D T cells exerted a differential interaction with the two CEA epitopes, i.e., displaying high affinity/CD8-independency toward the APL and low affinity/CD8-dependency toward the native CAP1 peptide. Our data demonstrate that the efficient detection of self-antigen expressed by tumors could be a feature of high avidity CD8-independent T cells, and underline the need for extensive analysis of tumor cross-recognition prior to any clinical usage of APL as anti-cancer vaccines.

Maximum likelihood Linear Programming Data Fusion for Speaker Recognition

Biometric system performance can be improved by means of data fusion. Several kinds of information can be fused in order to obtain a more accurate classification (identification or verification) of an input sample. In this paper we present a method for computing the weights in a weighted sum fusion for score combinations, by means of a likelihood model. The maximum likelihood estimation is set as a linear programming problem. The scores are derived from a GMM classifier working on a different feature extractor. Our experimental results assesed the robustness of the system in front a changes on time (different sessions) and robustness in front a change of microphone. The improvements obtained were significantly better (error bars of two standard deviations) than a uniform weighted sum or a uniform weighted product or the best single classifier. The proposed method scales computationaly with the number of scores to be fussioned as the simplex method for linear programming.

Application of the Mutual Information Minimization to speaker recognition / identification improvement

In this paper we propose the inversion of nonlinear distortions in order to improve the recognition rates of a speaker recognizer system. We study the effect of saturations on the test signals, trying to take into account real situations where the training material has been recorded in a controlled situation but the testing signals present some mismatch with the input signal level (saturations). The experimental results for speaker recognition shows that a combination of several strategies can improve the recognition rates with saturated test sentences from 80% to 89.39%, while the results with clean speech (without saturation) is 87.76% for one microphone, and for speaker identification can reduce the minimum detection cost function with saturated test sentences from 6.42% to 4.15%, while the results with clean speech (without saturation) is 5.74% for one microphone and 7.02% for the other one.

Hardy’s inequalities for monotone functions on partly ordered measure spaces

We characterize the weighted Hardy inequalities for monotone functions in Rn +. In dimension n = 1, this recovers the standard theory of Bp weights. For n > 1, the result was previously only known for the case p = 1. In fact, our main theorem is proved in the more general setting of partly ordered measure spaces.

Improving a leaves automatic recognition process using PCA

In this work we present a simulation of a recognition process with perimeter characterization of a simple plant leaves as a unique discriminating parameter. Data coding allowing for independence of leaves size and orientation may penalize performance recognition for some varieties. Border description sequences are then used, and Principal Component Analysis (PCA) is applied in order to study which is the best number of components for the classification task, implemented by means of a Support Vector Machine (SVM) System. Obtained results are satisfactory, and compared with [4] our system improves the recognition success, diminishing the variance at the same time.

Automatic Recognition of Leaves by Shape Detection Pre-Processing with Ica

In this work we present a simulation of a recognition process with perimeter characterization of a simple plant leaves as a unique discriminating parameter. Data coding allowing for independence of leaves size and orientation may penalize performance recognition for some varieties. Border description sequences are then used to characterize the leaves. Independent Component Analysis (ICA) is then applied in order to study which is the best number of components to be considered for the classification task, implemented by means of an Artificial Neural Network (ANN). Obtained results with ICA as a pre-processing tool are satisfactory, and compared with some references our system improves the recognition success up to 80.8% depending on the number of considered independent components.

Empirical mode decomposition-based face recognition system

In this work we explore the multivariate empirical mode decomposition combined with a Neural Network classifier as technique for face recognition tasks. Images are simultaneously decomposed by means of EMD and then the distance between the modes of the image and the modes of the representative image of each class is calculated using three different distance measures. Then, a neural network is trained using 10- fold cross validation in order to derive a classifier. Preliminary results (over 98 % of classification rate) are satisfactory and will justify a deep investigation on how to apply mEMD for face recognition.

Auditory localisation and recognition after left inferior collicular lesion: effects of work load on cortical activation patterns (fMRI study)

Evidence from neuropsychological and activation studies (Clarke et al., 2oo0, Maeder et al., 2000) suggests that sound recognitionand localisation are processed by two anatomically and functionally distinct cortical networks. We report here on a case of a patientthat had an interruption of auditory information and we show: i) the effects of this interruption on cortical auditory processing; ii)the effect of the workload on activation pattern.A 36 year old man suffered from a small left mesencephalic haemotrhage, due to cavernous angioma; the let% inferior colliculuswas resected in the surgical approach of the vascular malformation. In the acute stage, the patient complained of auditoryhallucinations and of auditory loss in right ear, while tonal audiometry was normal. At 12 months, auditory recognition, auditorylocalisation (assessed by lTD and IID cues) and auditory motion perception were normal (Clarke et al., 2000), while verbal dichoticlistening was deficient on the right side.Sound recognition and sound localisation activation patterns were investigated with fMRI, using a passive and an activeparadigm. In normal subjects, distinct cortical networks were involved in sound recognition and localisation, both in passive andactive paradigm (Maeder et al., 2OOOa, 2000b).Passive listening of environmental and spatial stimuli as compared to rest strongly activated right auditory cortex, but failed toactivate left primary auditory cortex. The specialised networks for sound recognition and localisation could not be visual&d onthe right and only minimally on the left convexity. A very different activation pattern was obtained in the active condition wherea motor response was required. Workload not only increased the activation of the right auditory cortex, but also allowed theactivation of the left primary auditory cortex. The specialised networks for sound recognition and localisation were almostcompletely present in both hemispheres.These results show that increasing the workload can i) help to recruit cortical region in the auditory deafferented hemisphere;and ii) lead to processing auditory information within specific cortical networks.References:Clarke et al. (2000). Neuropsychologia 38: 797-807.Mae.der et al. (2OOOa), Neuroimage 11: S52.Maeder et al. (2OOOb), Neuroimage 11: S33