CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Streptozotocin induces a shift towards T regulatory cell responses in vivo

Objectives: Streptozotocin (STZ) induced diabetes is currently the most commonly used animalmodel for islet transplantation.However, STZtreatment and the ensuing hyperglycemia were both shown to affect the immune response, including an apparent induction of lymphopenia. The aim of this study was to evaluate the respective effect of STZ and hyperglycemia on the immune system in STZ induced diabetic C57BL/6 mice. Methods: Phenotypes and levels of T and B cells were analyzed by flow cytometry in blood and spleen over time. The effect of hyperglycemia was further characterized by insulin replacement, islet transplantation and by using Rip (rat insulin promoter) DTR (dipheteria tocin receptor) transgenic mice. Results: STZ but not hyperglycemia was toxic for splenocytes in vitro, whereas hyperglycemia correlated with diabetes associated blood and spleen lymphopenia in vivo. Moreover, independently of hyperglycemia, STZ lead to a relative increase of T regulatory cells which retained their suppressive capacity in vitro. Conclusion: These data suggest thatSTZand the ensuing acute hyperglycemia have major direct and indirect effects on immune homeostasis. Thus, high caution needs to be exercised in the interpretation of the results of tolerance induction and/or immunosuppressive protocols in STZ-induced diabetes and islet transplantation models.

IL-10 controls cystatin C synthesis and blood concentration in response to inflammation through regulation of IFN regulatory factor 8 expression.

Cystatin C (CstC) is a cysteine protease inhibitor of major clinical importance. Low concentration of serum CstC is linked to atherosclerosis. CstC can prevent formation of amyloid β associated with Alzheimer's disease and can itself form toxic aggregates. CstC regulates NO secretion by macrophages and is a TGF-β antagonist. Finally, the serum concentration of CstC is an indicator of kidney function. Yet, little is known about the regulation of CstC expression in vivo. In this study, we demonstrate that the transcription factor IFN regulatory factor 8 (IRF-8) is critical for CstC expression in primary dendritic cells. Only those cells with IRF-8 bound to the CstC gene promoter expressed high levels of the inhibitor. Secretion of IL-10 in response to inflammatory stimuli downregulated IRF-8 expression and consequently CstC synthesis in vivo. Furthermore, the serum concentration of CstC decreased in an IL-10-dependent manner in mice treated with the TLR9 agonist CpG. CstC synthesis is therefore more tightly regulated than hitherto recognized. The mechanisms involved in this regulation might be targeted to alter CstC production, with potential therapeutic value. Our results also indicate that caution should be exerted when using the concentration of serum CstC as an indicator of kidney function in conditions in which inflammation may alter CstC production.

Transcriptional regulatory patterns of the myelin basic protein and malic enzyme genes by the thyroid hormone receptors alpha1 and beta1.

While there is evidence that the two ubiquitously expressed thyroid hormone (T3) receptors, TRalpha1 and TRbeta1, have distinct functional specificities, the mechanism by which they discriminate potential target genes remains largely unexplained. In this study, we demonstrate that the thyroid hormone response elements (TRE) from the malic enzyme and myelin basic protein genes (METRE and MBPTRE) respectively, are not functionally equivalent. The METRE, which is a direct repeat motif with a 4-base pair gap between the two half-site hexamers binds thyroid hormone receptor as a heterodimer with 9-cis-retinoic acid receptor (RXR) and mediates a high T3-dependent activation in response to TRalpha1 or TRbeta1 in NIH3T3 cells. In contrast, the MBPTRE, which consists of an inverted palindrome formed by two hexamers spaced by 6 base pairs, confers an efficient transactivation by TRbeta1 but a poor transactivation by TRalpha1. While both receptors form heterodimers with RXR on MBPTRE, the poor transactivation by TRalpha1 correlates also with its ability to bind efficiently as a monomer. This monomer, which is only observed with TRalpha1 bound to MBPTRE, interacts neither with N-CoR nor with SRC-1, explaining its functional inefficacy. However, in Xenopus oocytes, in which RXR proteins are not detectable, the transactivation mediated by TRalpha1 and TRbeta1 is equivalent and independent of a RXR supply, raising the question of the identity of the thyroid hormone receptor partner in these cells. Thus, in mammalian cells, the binding characteristics of TRalpha1 to MBPTRE (i.e. high monomer binding efficiency and low transactivation activity) might explain the particular pattern of T3 responsiveness of MBP gene expression during central nervous system development.

cis- and trans-acting elements involved in reactivation of vaccinia virus early transcription.

We have previously shown that transcription from the vaccinia virus 7.5K early promoter is reactivated late in infection (J. Garcés, K. Masternak, B. Kunz, and R. Wittek, J. Virol. 67:5394-5401, 1993). To identify the sequence elements mediating reactivation, we constructed recombinant viruses harboring deletions, substitutions, or insertions in the 7.5K promoter or its flanking regions. The analysis of these viruses showed that sequences both upstream as well as downstream of the transcription initiation site contribute to reactivation of the 7.5K promoter. We tested whether reactivation could be explained by a high affinity of vaccinia virus early transcription factor to reactivated promoters. Bandshift experiments using purified protein showed that promoters which bind the factor with high affinity in general also have high early transcriptional activity. However, no correlation was found between affinity of the factor and reactivation. Interestingly, overexpression of recombinant early transcription factor in vaccinia virus-infected cells resulted in a shutdown of late transcription and in reactivation of promoters, which are normally not reactivated.

cis- and trans-acting elements of the estrogen-regulated vitellogenin gene B1 of Xenopus laevis.

Vitellogenin genes are expressed under strict estrogen control in the liver of female oviparous vertebrates. Gene transfer experiments using estrogen-responsive cells have shown that the 13 bp perfect palindromic element GGTCACTGTGACC found upstream of the Xenopus laevis vitellogenin gene A2 promoter mediates hormonal stimulation and thus, was called the estrogen-responsive element (ERE). In the Xenopus vitellogenin genes B1 and B2 there are two closely adjacent EREs with one or more base substitutions when compared to the consensus ERE GGTCANNNTGACC. On their own, these degenerated elements have only a low or no regulatory capacity at all but act together synergistically to form an estrogen-responsive unit (ERU) with the same strength as the perfect palindromic 13 bp element. Analysis of estrogen receptor binding to the gene B1 ERU revealed a cooperative interaction of receptor dimers to the two adjacent imperfect EREs which most likely explains the synergistic stimulation observed in vivo. Furthermore, a promoter activator element located between positions --113 and --42 of the gene B1 and functional in the human MCF-7 and the Xenopus B3.2 cells has been identified and shown to be involved in the high level of induced transcription activity when the ERE is placed at a distance from the promoter. Finally, a hormone-controlled in vitro transcription system derived from Xenopus liver nuclear extracts was exploited to characterize two additional novel cis-acting elements within the vitellogenin gene B1 promoter. One of them, a negative regulatory element (NRE), is responsible for repression of promoter activity in the absence of hormone. The second is related to the NF-I binding site and is required, together with the ERE, to mediate hormonal induction. Moreover, we detected three trans-acting activities in Xenopus liver nuclear extracts that interact with these regions and demonstrated that they participate in the regulation of the expression of the vitellogenin promoter in vitro.

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining tilling mutant populations.

Background: TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results: Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions: This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation.

Regulatory roles of microtubule-associated proteins in neuronal morphogenesis. Involvement of the extracellular matrix

As a result of recent investigations, the cytoskeleton can be viewed as a cytoplasmic system of interconnected filaments with three major integrative levels: self-assembling macromolecules, filamentous polymers, e.g., microtubules, intermediate filaments and actin filaments, and supramolecular structures formed by bundles of these filaments or networks resulting from cross-bridges between these major cytoskeletal polymers. The organization of this biological structure appears to be sensitive to fine spatially and temporally dependent regulatory signals. In differentiating neurons, regulation of cytoskeleton organization is particularly relevant, and the microtubule-associated protein (MAP) tau appears to play roles in the extension of large neuritic processes and axons as well as in the stabilization of microtubular polymers along these processes. Within this context, tau is directly involved in defining neuronal polarity as well as in the generation of neuronal growth cones. There is increasing evidence that elements of the extracellular matrix contribute to the control of cytoskeleton organization in differentiating neurons, and that these regulations could be mediated by changes in MAP activity. In this brief review, we discuss the possible roles of tau in mediating the effects of extracellular matrix components on the internal cytoskeletal arrays and its organization in growing neurons.

Influence of the polymorphisms of the α-major regulatory element HS-40 on in vitro gene expression

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

Lack of evidence for regulation of cardiac P-type ATPases and MAP kinases in transgenic mice with cardiac-specific overexpression of constitutively active α1B-adrenoceptors

The regulatory function of &#945;1B-adrenoceptors in mammalian heart homeostasis is controversial. The objective of the present study was to characterize the expression/activity of key proteins implicated in cardiac calcium handling (Na+/K+-ATPase and Ca2+-ATPases) and growth (ERK1/2, JNK1/2 and p38) in mice with cardiac-selective overexpression of constitutively active mutant &#945;1B-adrenoceptor (CAM&#945;1B-AR), which present a mild cardiac hypertrophy phenotype. Immunoblot assays showed that myocardial plasma membrane Ca2+-ATPase (PMCA) expression was increased by 30% in CAM&#945;1B-AR mice (N = 6, P < 0.05), although there was no change in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) expression. Moreover, total Ca2+-ATPase activity was not modified, but a significant increase in the activity of the thapsigargin-resistant (PMCA) to thapsigargin-sensitive (SERCA) ratio was detected. Neither Na+/K+-ATPase activity nor the expression of &#945;1 and &#945;2 subunit isoforms was changed in CAM&#945;1B-AR mouse hearts. Moreover, immunoblot assays did not provide evidence for an enhanced activation of the three mitogen-activated protein kinases studied in this stage of hypertrophy. Therefore, these findings indicate that chronic cardiac &#945;1B-AR activation in vivo led to mild hypertrophy devoid of significant signs of adaptive modifications concerning primary intracellular calcium control and growth-related proteins, suggesting a minor pathophysiological role of this adrenergic receptor in mouse heart at this stage of development.

Critical elements underpinning the emergence of the medical game ecosystem: gamifying traumatic brain injury rehabilitation in Finland

The healthcare sector is currently in the verge of a reform and thus, the medical game research provide an interesting area of research. The aim of this study is to explore the critical elements underpinning the emergence of the medical game ecosystem with three sub-objectives: (1) to seek who are the key actors involved in the medical game ecosystem and identify their needs, (2) to scrutinise what types of resources are required in medical game development and what types of relationships are needed to secure those resources, and (3) to identify the existing institutions (‘the rules of the game’) affecting the emergence of the medical game ecosystem. The theoretical background consists of service ecosystems literature. The empirical study conducted is based on the semi-structured theme interviews of 25 experts in three relevant fields: games and technology, health and funding. The data was analysed through a theoretical framework that was designed based upon service ecosystems literature. The study proposes that the key actors are divided into five groups: medical game companies, customers, funders, regulatory parties and complementors. Their needs are linked to improving patient motivation and enhancing the healthcare processes resulting in lower costs. Several types of resources, especially skills and knowledge, are required to create a medical game. To gain access to those resources, medical game companies need to build complex networks of relationships. Proficiency in managing those value networks is crucial. In addition, the company should take into account the underlying institutions in the healthcare sector affecting the medical game ecosystem. Three crucial institutions were identified: validation, lack of innovation supporting structures in healthcare and the rising consumerisation. Based on the findings, medical games cannot be made in isolation. A developmental trajectory model of the emerging medical game ecosystem was created based on the empirical data. The relevancy of relationships and resources is dependent on the trajectory that the medical game company at that time resides. Furthermore, creating an official and documented database for clinically valdated medical games was proposed to establish the medical game market and ensure an adequate status for the effective medical games. Finally, ecosystems approach provides interesting future opportunities for research on medical game ecosystems.

Critical elements underpinning the emergence of the medical game ecosystem:gamifying traumatic brain injury rehabilitation in Finland

The healthcare sector is currently in the verge of a reform and thus, the medical game research provide an interesting area of research. The aim of this study is to explore the critical elements underpinning the emergence of the medical game ecosystem with three sub-objectives: (1) to seek who are the key actors involved in the medical game ecosystem and identify their needs, (2) to scrutinise what types of resources are required in medical game development and what types of relationships are needed to secure those resources, and (3) to identify the existing institutions (‘the rules of the game’) affecting the emergence of the medical game ecosystem. The theoretical background consists of service ecosystems literature. The empirical study conducted is based on the semi-structured theme interviews of 25 experts in three relevant fields: games and technology, health and funding. The data was analysed through a theoretical framework that was designed based upon service ecosystems literature. The study proposes that the key actors are divided into five groups: medical game companies, customers, funders, regulatory parties and complementors. Their needs are linked to improving patient motivation and enhancing the healthcare processes resulting in lower costs. Several types of resources, especially skills and knowledge, are required to create a medical game. To gain access to those resources, medical game companies need to build complex networks of relationships. Proficiency in managing those value networks is crucial. In addition, the company should take into account the underlying institutions in the healthcare sector affecting the medical game ecosystem. Three crucial institutions were identified: validation, lack of innovation supporting structures in healthcare and the rising consumerisation. Based on the findings, medical games cannot be made in isolation. A developmental trajectory model of the emerging medical game ecosystem was created based on the empirical data. The relevancy of relationships and resources is dependent on the trajectory that the medical game company at that time resides. Furthermore, creating an official and documented database for clinically validated medical games was proposed to establish the medical game market and ensure an adequate status for the effective medical games. Finally, ecosystems approach provides interesting future opportunities for research on medical game ecosystems

Role of the homeodomain transcription factor Hoxa13 in embryonic development and formation of extra-embryonic structures

La famille des gènes Hox code pour des facteurs de transcription connus pour leur contribution essentielle à l’élaboration de l’architecture du corps et ce, au sein de tout le règne animal. Au cours de l’évolution chez les vertébrés, les gènes Hox ont été redéfinis pour générer toute une variété de nouveaux tissus/organes. Souvent, cette diversification s’est effectuée via des changements quant au contrôle transcriptionnel des gènes Hox. Chez les mammifères, la fonction de Hoxa13 n’est pas restreinte qu’à l’embryon même, mais s’avère également essentielle pour le développement de la vascularisation fœtale au sein du labyrinthe placentaire, suggérant ainsi que sa fonction au sein de cette structure aurait accompagné l’émergence des espèces placentaires. Au chapitre 2, nous mettons en lumière le recrutement de deux autres gènes Hoxa, soient Hoxa10 et Hoxa11, au compartiment extra-embryonnaire. Nous démontrons que l’expression de Hoxa10, Hoxa11 et Hoxa13 est requise au sein de l’allantoïde, précurseur du cordon ombilical et du système vasculaire fœtal au sein du labyrinthe placentaire. De façon intéressante, nous avons découvert que l’expression des gènes Hoxa10-13 dans l’allantoïde n’est pas restreinte qu’aux mammifères placentaires, mais est également présente chez un vertébré non-placentaire, indiquant que le recrutement des ces gènes dans l’allantoïde précède fort probablement l’émergence des espèces placentaires. Nous avons généré des réarrangements génétiques et utilisé des essais transgéniques pour étudier les mécanismes régulant l’expression des gènes Hoxa dans l’allantoïde. Nous avons identifié un fragment intergénique de 50 kb capable d’induire l’expression d’un gène rapporteur dans l’allantoïde. Cependant, nous avons trouvé que le mécanisme de régulation contrôlant l’expression du gène Hoxa au sein du compartiment extra-embryonnaire est fort complexe et repose sur plus qu’un seul élément cis-régulateur. Au chapitre 3, nous avons utilisé la cartographie génétique du destin cellulaire pour évaluer la contribution globale des cellules exprimant Hoxa13 aux différentes structures embryonnaires. Plus particulièrement, nous avons examiné plus en détail l’analyse de la cartographie du destin cellulaire de Hoxa13 dans les pattes antérieures en développement. Nous avons pu déterminer que, dans le squelette du membre, tous les éléments squelettiques de l’autopode (main), à l’exception de quelques cellules dans les éléments carpiens les plus proximaux, proviennent des cellules exprimant Hoxa13. En contraste, nous avons découvert que, au sein du compartiment musculaire, les cellules exprimant Hoxa13 et leurs descendantes (Hoxa13lin+) s’étendent à des domaines plus proximaux du membre, où ils contribuent à générer la plupart des masses musculaires de l’avant-bras et, en partie, du triceps. De façon intéressante, nous avons découvert que les cellules exprimant Hoxa13 et leurs descendantes ne sont pas distribuées uniformément parmi les différents muscles. Au sein d’une même masse musculaire, les fibres avec une contribution Hoxa13lin+ différente peuvent être identifiées et les fibres avec une contribution semblable sont souvent regroupées ensemble. Ce résultat évoque la possibilité que Hoxa13 soit impliqué dans la mise en place de caractéristiques spécifiques des groupes musculaires, ou la mise en place de connections nerf-muscle. Prises dans leur ensemble, les données ici présentées permettent de mieux comprendre le rôle de Hoxa13 au sein des compartiments embryonnaires et extra-embryonnaires. Par ailleurs, nos résultats seront d’une importance primordiale pour soutenir les futures études visant à expliquer les mécanismes transcriptionnels soutenant la régulation des gènes Hoxa dans les tissus extra-embryonnaires.

Global transcript analysis of rice leaf and seed using SAGE technology.

We have compiled two comprehensive gene expression profiles from mature leaf and immature seed tissue of rice (Oryza sativa ssp. japonica cultivar Nipponbare) using Serial Analysis of Gene Expression (SAGE) technology. Analysis revealed a total of 50 519 SAGE tags, corresponding to 15 131 unique transcripts. Of these, the large majority (approximately 70%) occur only once in both libraries. Unexpectedly, the most abundant transcript (approximately 3% of the total) in the leaf library was derived from a type 3 metallothionein gene. The overall frequency profiles of the abundant tag species from both tissues differ greatly and reveal seed tissue as exhibiting a non-typical pattern of gene expression characterized by an over abundance of a small number of transcripts coding for storage proteins. A high proportion ( approximately 80%) of the abundant tags (> or = 9) matched entries in our reference rice EST database, with many fewer matches for low abundant tags. Singleton transcripts that are common to both tissues were collated to generate a summary of low abundant transcripts that are expressed constitutively in rice tissues. Finally and most surprisingly, a significant number of tags were found to code for antisense transcripts, a finding that suggests a novel mechanism of gene regulation, and may have implications for the use of antisense constructs in transgenic technology.