Cre recombinase-mediated inactivation of H-2Dd transgene expression: evidence for partial missing self-recognition by Ly49A NK cells.

We have established H-2D(d)-transgenic (Tg) mice, in which H-2D(d) expression can be extinguished by Cre recombinase-mediated deletion of an essential portion of the transgene (Tg). NK cells adapted to the expression of the H-2D(d) Tg in H-2(b) mice and acquired reactivity to cells lacking H-2D(d), both in vivo and in vitro. H-2D(d)-Tg mice crossed to mice harboring an Mx-Cre Tg resulted in mosaic H-2D(d) expression. That abrogated NK cell reactivity to cells lacking D(d). In D(d) single Tg mice it is the Ly49A+ NK cell subset that reacts to cells lacking D(d), because the inhibitory Ly49A receptor is no longer engaged by its D(d) ligand. In contrast, Ly49A+ NK cells from D(d) x MxCre double Tg mice were unable to react to D(d)-negative cells. These Ly49A+ NK cells retained reactivity to target cells that were completely devoid of MHC class I molecules, suggesting that they were not anergic. Variegated D(d) expression thus impacts specifically missing D(d) but not globally missing class I reactivity by Ly49A+ NK cells. We propose that the absence of D(d) from some host cells results in the acquisition of only partial missing self-reactivity.

Education of Murine NK Cells Requires Both cis and trans Recognition of MHC Class I Molecules.

Although NK cells use invariant receptors to identify diseased cells, they nevertheless adapt to their environment, including the presence of certain MHC class I (MHC-I) molecules. This NK cell education, which is mediated by inhibitory receptors specific for MHC-I molecules, changes the responsiveness of activating NK cell receptors (licensing) and modifies the repertoire of MHC-I receptors used by NK cells. The fact that certain MHC-I receptors have the unusual capacity to recognize MHC-I molecules expressed by other cells (trans) and by the NK cell itself (cis) has raised the question regarding possible contributions of the two types of interactions to NK cell education. Although the analysis of an MHC-I receptor variant suggested a role for cis interaction for NK cell licensing, adoptive NK cell transfer experiments supported a key role for trans recognition. To reconcile some of these findings, we have analyzed the impact of cell type-specific deletion of an MHC-I molecule and of a novel MHC-I receptor variant on the education of murine NK cells when these mature under steady-state conditions in vivo. We find that MHC-I expression by NK cells (cis) and by T cells (trans), and MHC-I recognition in cis and in trans, are both needed for NK cell licensing. Unexpectedly, modifications of the MHC-I receptor repertoire are chiefly dependent on cis binding, which provides additional support for an essential role for this unconventional type of interaction for NK cell education. These data suggest that two separate functions of MHC-I receptors are needed to adapt NK cells to self-MHC-I.

Intracellular pattern-recognition receptors

The last ten years of research in the field of innate immunity have been incredibly fertile: the transmembrane Toll-like receptors (TLRs) were discovered as guardians protecting the host against microbial attacks and the emerging pathways characterized in detail. More recently, cytoplasmic sensors were identified, which are capable of detecting not only microbial, but also self molecules. Importantly, while such receptors trigger crucial host responses to microbial insult, over-activity of some of them has been linked to autoinflammatory disorders, hence demonstrating the importance of tightly regulating their actions over time and space. Here, we provide an overview of recent findings covering this area of innate and inflammatory responses that originate from the cytoplasm

Theoretical study of the electrostatically driven step of receptor-G protein recognition.

This study proposes a theoretical model describing the electrostatically driven step of the alpha 1 b-adrenergic receptor (AR)-G protein recognition. The comparative analysis of the structural-dynamics features of functionally different receptor forms, i.e., the wild type (ground state) and its constitutively active mutants D142A and A293E, was instrumental to gain insight on the receptor-G protein electrostatic and steric complementarity. Rigid body docking simulations between the different forms of the alpha 1 b-AR and the heterotrimeric G alpha q, G alpha s, G alpha i1, and G alpha t suggest that the cytosolic crevice shared by the active receptor and including the second and the third intracellular loops as well as the cytosolic extension of helices 5 and 6, represents the receptor surface with docking complementarity with the G protein. On the other hand, the G protein solvent-exposed portions that recognize the intracellular loops of the activated receptors are the N-terminal portion of alpha 3, alpha G, the alpha G/alpha 4 loop, alpha 4, the alpha 4/beta 6 loop, alpha 5, and the C-terminus. Docking simulations suggest that the two constitutively active mutants D142A and A293E recognize different G proteins with similar selectivity orders, i.e., G alpha q approximately equal to G alpha s > G alpha i > G alpha t. The theoretical models herein proposed might provide useful suggestions for new experiments aiming at exploring the receptor-G protein interface.

Phylogeny and circumscription of Sapindaceae revisited: molecular sequence data, morphology and biogeography support recognition of a new family, Xanthoceraceae

Background and aims Recent studies have adopted a broad definition of Sapindaceae that includes taxa traditionally placed in Aceraceae and Hippocastanaceae, achieving monophyly but yielding a family difficult to characterize and for which no obvious morphological synapomorphy exists. This expanded circumscription was necessitated by the finding that the monotypic, temperate Asian genus Xanthoceras, historically placed in Sapindaceae tribe Harpullieae, is basal within the group. Here we seek to clarify the relationships of Xanthoceras based on phylogenetic analyses using a dataset encompassing nearly 3/4 of sapindaceous genera, comparing the results with information from morphology and biogeography, in particular with respect to the other taxa placed in Harpullieae. We then re-examine the appropriateness of maintaining the current broad, morphologically heterogeneous definition of Sapindaceae and explore the advantages of an alternative family circumscription. Methods Using 243 samples representing 104 of the 142 currently recognized genera of Sapindaceae s. lat. (including all in Harpullieae), sequence data were analyzed for nuclear (ITS) and plastid (matK, rpoB, trnD-trnT, trnK-matK, trnL-trnF and trnS-trnG) markers, adopting the methodology of a recent family-wide study, performing single-gene and total evidence analyses based on maximum likelihood (ML) and maximum parsimony (MP) criteria, and applying heuristic searches developed for large datasets, viz, a new strategy implemented in RAxML (for ML) and the parsimony ratchet (for MP). Bootstrap analyses were performed for each method to test for congruence between markers. Key results Our findings support earlier suggestions that Harpullieae are polyphyletic: Xanthoceras is confirmed as sister to all other sampled taxa of Sapindaceae s. lat.; the remaining members belong to three other clades within Sapindaceae s. lat., two of which correspond respectively to the groups traditionally treated as Aceraceae and Hippocastanaceae, together forming a clade sister to the largely tropical Sapindaceae s. str., which is monophyletic and morphologically coherent provided Xanthoceras is excluded. Conclusion To overcome the difficulties of a broadly circumscribed Sapindaceae, we resurrect the historically recognized temperate families Aceraceae and Hippocastanaceae, and describe a new family, Xanthoceraceae, thus adopting a monophyletic and easily characterized circumscription of Sapindaceae nearly identical to that used for over a century.

Cluster recognition in spatial-temporal sequences: The case of forest fires

Forest fire sequences can be modelled as a stochastic point process where events are characterized by their spatial locations and occurrence in time. Cluster analysis permits the detection of the space/time pattern distribution of forest fires. These analyses are useful to assist fire-managers in identifying risk areas, implementing preventive measures and conducting strategies for an efficient distribution of the firefighting resources. This paper aims to identify hot spots in forest fire sequences by means of the space-time scan statistics permutation model (STSSP) and a geographical information system (GIS) for data and results visualization. The scan statistical methodology uses a scanning window, which moves across space and time, detecting local excesses of events in specific areas over a certain period of time. Finally, the statistical significance of each cluster is evaluated through Monte Carlo hypothesis testing. The case study is the forest fires registered by the Forest Service in Canton Ticino (Switzerland) from 1969 to 2008. This dataset consists of geo-referenced single events including the location of the ignition points and additional information. The data were aggregated into three sub-periods (considering important preventive legal dispositions) and two main ignition-causes (lightning and anthropogenic causes). Results revealed that forest fire events in Ticino are mainly clustered in the southern region where most of the population is settled. Our analysis uncovered local hot spots arising from extemporaneous arson activities. Results regarding the naturally-caused fires (lightning fires) disclosed two clusters detected in the northern mountainous area.

Characterization of the immunological properties of " Plasmodium falciparum " and " Plasmodium berghei " circumsporozoite proteins : antibody responses and recognition by specific CD8+ T cells

SUMMARY Interest in developing intervention strategies against malaria by targeting the liver stage of the Plasmodium life cycle has been fueled by studies which show that sterile protective immunity can be achieved by immunization with radiation-attenuated sporozoites. Anti-malarial drugs and insecticides have been widely used to control the disease, but in the hope of developing a more cost-effective intervention strategy, vaccine development has taken centre stage in malaria research. There is currently no vaccine against malaria. Attenuated sporozoite-induced immunity is achieved by antibodies and T cells against malaria liver stage antigens, the most abundant being the circumsporozoite protein (CSP), and many vaccine formulations aim at mimicking this immunity. However, the mechanisms by which the antibody and T cell immune responses are generated after infection by sporozoites, or after immunization with different vaccine formulations are still not well understood. The first part of this work aimed at determining the ability of primary hepatocytes from BALB/c mice to process and present CSP-derived peptides after infection with P. berghei sporozoites. Both infected hepatocytes and those traversed by sporozoites during migration were found to be capable of processing and presenting the CSP to specific CD8+ T cells in vitro. The pathway of processing and presentation involved the proteasome, aspartic proteases and transport through a post-Endoplasmic Reticulum (ER) compartment. These results suggest that in vivo, infected hepatocytes contribute to the elicitation and expansion of a T cell response. In the second part, the antibody responses of CB6F1 mice to synthetic peptides corresponding to the N- and C-terminal domains of P. berghei and P. falciparum CS proteins were characterized. Mice were immunized with single peptides or a combination of N- and C-terminal peptides. The peptides were immunogenic in mice and the antisera generated could recognize the native CSP on the sporozoite surface. Antisera generated against the N-terminal peptides or against the combinations inhibited sporozoite invasion of hepatocytes in vitro. In vivo, more mice immunized with single P. berghei peptides were protected from infection upon a challenge with P. berghei sporozoites, than mice immunized with a combination of N- and C-terminal peptides. Furthermore, P. falciparum N-terminal peptides were recognized by serum samples from people living in malaria-endemic areas. Importantly, recognition of a peptide from the N-terminal fragment of the P. falciparum CSP by sera from children living in a malaria-endemic region was associated with protection from disease. These results underline the potential of using such peptides as malaria vaccine candidates. RESUME L'intérêt de développer des stratégies d'intervention contre la malaria ciblant le stade pré-erythrocytaire a été alimenté par des études qui montrent qu'il est possible d'obtenir une immunité par l'injection de sporozoites irradiés. Les médicaments et les insecticides anti-paludiques ont été largement utilisés pour contrôler la maladie, mais dans l'espoir de développer une stratégie d'intervention plus rentable, le développement de vaccins a été placé au centre des recherches actuelles contre la malaria. A l'heure actuelle, il n'existe aucun vaccin contre la malaria. L'immunité induite par les sporozoites irradiés est due à l'effet combiné d'anticorps et de cellules T qui agissent contre les antigènes du stade hépatique dont le plus abondant est la protéine circumsporozoite (CSP). Beaucoup de formulations de vaccin visent à imiter l'immunité induite par les sporozoites irradiés. Cependant, les mécanismes par lesquels les anticorps et les cellules T sont génerés après infection par les sporozoites ou après immunisation avec des formulations de vaccin ne sont pas bien compris. La première partie de ce travail a visé à déterminer la capacité de hépatocytes primaires provenant de souris BALB/c à "processer" et à présenter des peptides dérivés de la CSP, après infection par des sporozoites de Plasmodium berghei. Nous avons montré que in vitro, les hépatocytes infectés et ceux traversés par les sporozoites pendant leur migration étaient capables de "processer" et de présenter la CSP aux cellules T CD8+ spécifiques. La voie de présentation implique le protéasome, les protéases de type aspartique et le transport à travers un compartiment post-reticulum endoplasmique. Ces résultats suggèrent que in vivo, les hépatocytes infectés contribuent à l'induction et à l'expansion d'une réponse immunitaire spécifique aux cellules T. Dans la deuxième partie, nous avons caractérisé les réponses anticorps chez les souris de la souche CB6F1 face aux peptides N- et C-terminaux des protéines circumsporozoites de Plasmodium berghei et Plasmodium falciparum. Les souris ont été immunisées avec les peptides individuellement ou en combinaison. Les peptides utilisés étaient immunogéniques chez les souris, et les anticorps produits pouvaient reconnaître la protéine CSP native à la surface des sporozoites. In vitro, les sera contre les peptides N-teminaux et les combinaisons étaient capables d'inhiber l'invasion de hépatocytes par les sporozoites. In vivo, plus de souris immunisées avec les peptides individuels de la CSP de P. berghei étaient protégées contre la malaria que les souris immunisées avec une combinaison de peptides N- et C-terminaux. De plus, les peptides N-terminaux de la CSP de P. falciparum ont été reconnus par les sera de personnes vivant dans des régions endémiques pour la malaria. Il est intéressant de voir que la reconnaissance d'un peptide N-terminal de P. falciparum par des sera d'enfants habitant dans des régions endémiques était associé à la protection contre la maladie. Ces résultats soulignent le potentiel de ces peptides comme candidats-vaccin contre la malaria.

Pattern recognition of sleep in rodents using piezoelectric signals generated by gross body movements.

Current research on sleep using experimental animals is limited by the expense and time-consuming nature of traditional EEG/EMG recordings. We present here an alternative, noninvasive approach utilizing piezoelectric films configured as highly sensitive motion detectors. These film strips attached to the floor of the rodent cage produce an electrical output in direct proportion to the distortion of the material. During sleep, movement associated with breathing is the predominant gross body movement and, thus, output from the piezoelectric transducer provided an accurate respiratory trace during sleep. During wake, respiratory movements are masked by other motor activities. An automatic pattern recognition system was developed to identify periods of sleep and wake using the piezoelectric generated signal. Due to the complex and highly variable waveforms that result from subtle postural adjustments in the animals, traditional signal analysis techniques were not sufficient for accurate classification of sleep versus wake. Therefore, a novel pattern recognition algorithm was developed that successfully distinguished sleep from wake in approximately 95% of all epochs. This algorithm may have general utility for a variety of signals in biomedical and engineering applications. This automated system for monitoring sleep is noninvasive, inexpensive, and may be useful for large-scale sleep studies including genetic approaches towards understanding sleep and sleep disorders, and the rapid screening of the efficacy of sleep or wake promoting drugs.

Community introduction of practice parameters for autistic spectrum disorders: Advancing early recognition.

OBJECTIVES: Within a strong interdisciplinary framework, improvement in the quality of care for children with autistic spectrum disorders through a 2 year implementation program of Practice Parameters, aimed principally at improving early detection and intervention. METHOD: We developed Practice Parameters (PPs) for Pervasive Developmental Disorders and circulated the PPs to all child and adolescent psychiatrists practicing in the region. RESULTS: PP development and parallel information strategies resulted in a significant decrease of 1.5 years in the mean-age-at-diagnosis. However, further analysis indicated that improvement was only transient. CONCLUSION: Despite the encouraging improvement in mean-age-at-diagnosis 2 years after PP implementation, other indicators showed a failure to maintain the improvements. A systematic screening program would be the most reliable method to reinforce the PPs.

Decreased specific CD8+ T cell cross-reactivity of antigen recognition following vaccination with Melan-A peptide.

The aim of T cell vaccines is the expansion of antigen-specific T cells able to confer immune protection against pathogens or tumors. Although increase in absolute cell numbers, effector functions and TCR repertoire of vaccine-induced T cells are often evaluated, their reactivity for the cognate antigen versus their cross-reactive potential is rarely considered. In fact, little information is available regarding the influence of vaccines on T cell fine specificity of antigen recognition despite the impact that this feature may have in protective immunity. To shed light on the cross-reactive potential of vaccine-induced cells, we analyzed the reactivity of CD8(+) T cells following vaccination of HLA-A2(+) melanoma patients with Melan-A peptide, incomplete Freund's adjuvant and CpG-oligodeoxynucleotide adjuvant, which was shown to induce strong expansion of Melan-A-reactive CD8(+) T cells in vivo. A collection of predicted Melan-A cross-reactive peptides, identified from a combinatorial peptide library, was used to probe functional antigen recognition of PBMC ex vivo and Melan-A-reactive CD8(+) T cell clones. While Melan-A-reactive CD8(+) T cells prior to vaccination are usually constituted of widely cross-reactive naive cells, we show that peptide vaccination resulted in expansion of memory T cells displaying a reactivity predominantly restricted to the antigen of interest. Importantly, these cells are tumor-reactive.

Enjeux éthiques des nouvelles technologies de la reproduction : une conception de la justice à l'interface de l'éthique du "care" et de l'approche des "capabilités"

L'émergence des nouvelles technologies de la reproduction (NTR) est allée de pair avec un certain nombre de discours. Un discours promettant d'une part une extension de la palette de choix reproductifs des individus, une extension de leur liberté et de leur autonomie reproductives, dont la forme la plus extrême peut se traduire par la formule : un enfant quand je veux et comme je veux. D'autre part, un discours annonçant une série de « catastrophes » à venir, telles que l'effondrement de l'institution de la famille et la modification de l'espèce humaine. En d'autres termes, une tension entre promesses et catastrophes qui place les sociétés contemporaines face à de nombreux défis sociaux, politiques et éthiques, notamment quant à la question de la régulation de la PMA (procréation médicalement assistée) : qui peut y avoir accès ? Quelles techniques doit-on autoriser ? Ou au contraire limiter ? Tant de questions auxquelles aucune réponse simple et évidente n'existe. La diversité des réponses législatives quant à ces questions illustre cette complexité. L'éthique peut, ici, jouer un rôle fondamental. Sans toutefois prétendre donner des réponses toutes faites et facilement applicables, elle offre un espace de réflexion, le privilège de prendre une certaine distance face à des enjeux contemporains. C'est dans cette perspective que nous avons ancré ce travail de recherche en questionnant les enjeux éthiques de la PMA à partir d'une perspective de justice. Toutefois, au sein des études en bioéthique, majoritairement issues de la tradition libérale, la tension énoncée précédemment mène la bioéthique à justifier un certain nombre d'inégalités plutôt que de veiller à les dépasser. Ainsi, une évaluation de la pratique de la PMA à partir d'une perspective de la justice, exige, au préalable, une réévaluation du concept même de justice. Ce faisant, par une articulation entre l'éthique du care de Joan Tronto et l'approche des capabilités de Martha Nussbaum qui placent la vulnérabilité au coeur de la personne, nous avons proposé une conception de la justice fondée sur une anthropologie de la vulnérabilité. Cette conception nous permet d'identifier, dans le cadre de la pratique de la PMA en Suisse et en partant de la loi sur la procréation assistée (LPMA), les constructions normatives qui mènent à la non-reconnaissance et, ce faisant, à la mise à l'écart, de certaines formes de vulnérabilité : une vulnérabilité générique et une vulnérabilité socio-économique. Traitant la question de la vulnérabilité générique principalement, nos analyses ont une incidence sur les conceptions de la famille, du bien de l'enfant, de la femme et de la nature, telles qu'elles sont actuellement véhiculées par une conception naturalisée de la PMA. Répondre aux vulnérabilités identifiées, en veillant à leur donner une place, signifie alors déplacer ces conceptions naturalisées, afin que les vulnérabilités soient intégrées aux pratiques sociales et que les exigences de justice soient ainsi remplies. - The emergence of assisted reproductive technologies (ART) came along with several discourses. On the one hand a discourse promising an extension of the individuals' reproductive choices, their procreative liberty and autonomy. On the other hand a discourse announced a series of disasters to come such as the collapse of the family institution and the modification of human kind. In other words, a growing tension appears between promises and disasters and contemporary societies are facing inevitable social, political and ethical challenges, in particular with regard to the issue of ART regulation: who has access? What procedures should be authorized? Which ones should be limited? These complex questions have no simple or obvious answers. The variety of legislative responses to these questions highlights complexity. Ethics can play a fundamental role, and without claiming to give simple answers, also offer a space for reflection as well as the privilege to distance itself with regard to contemporary issues. It is in this perspective that this study questions the ethical considerations of ART in a perspective of justice. However, in previous studies in bioethics mainly following a liberal tradition, previously mentioned tension has lead bioethics to justify some inequalities instead of trying to overcome them. As a consequence, evaluating practices of ART from a perspective of justice requires to first reevaluate the concept of justice itself. In doing so we offer a conception of justice founded on the anthropology of vulnerability. This conception draws on an articulation of the ethic of care of Joan Tronto and the capability approach of Martha Nussbaum, which places vulnerability at the center of the person. This conception allows us to identify, within the framework of ARTS in Switzerland and starting with the laws of medically assisted procreation (LPMA), some normative constructions. These constructions lead to the non-recognition and the disregard of some forms of vulnerability: a generic vulnerability as well as socio-economic counterpart. Focusing mainly on the issue of generic vulnerability, our analysis has implications for the conceptions of family, the best interests of the child, woman, and nature in the way they are defined in a naturalized conception of ART. Responding to such failures by taking into account these vulnerabilities thus means to move these conceptions in order for vulnerabilities to be integrated in social practices and requirements for justice to be fulfilled.

The efficiency of antigen recognition by CD8+ CTL clones is determined by the frequency of serial TCR engagement.

Using H-2Kd-restricted CTL clones, which are specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS(252-260) (SYIPSAEKI) and permit assessment of TCR-ligand interactions by TCR photoaffinity labeling, we have previously identified several peptide derivative variants for which TCR-ligand binding and the efficiency of Ag recognition deviated by fivefold or more. Here we report that the functional CTL response (cytotoxicity and IFN-gamma production) correlated with the rate of TCR-ligand complex dissociation, but not the avidity of TCR-ligand binding. While peptide antagonists exhibited very rapid TCR-ligand complex dissociation, slightly slower dissociation was observed for strong agonists. Conversely and surprisingly, weak agonists typically displayed slower dissociation than the wild-type agonists. Acceleration of TCR-ligand complex dissociation by blocking CD8 participation in TCR-ligand binding increased the efficiency of Ag recognition in cases where dissociation was slow. In addition, permanent TCR engagement by TCR-ligand photocross-linking completely abolished sustained intracellular calcium mobilization, which is required for T cell activation. These results indicate that the functional CTL response depends on the frequency of serial TCR engagement, which, in turn, is determined by the rate of TCR-ligand complex dissociation.