Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14.

Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage. LBP has been shown previously to potentiate the host response to LPS. However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results. Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS. Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia. Three classes of mAbs were obtained. Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity. In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia. These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity.

Genetic diversity and differentiation processes in the ploidy series of Olea europaea L.: a multiscale approach from subspecies to insular populations.

Geographical isolation and polyploidization are central concepts in plant evolution. The hierarchical organization of archipelagos in this study provides a framework for testing the evolutionary consequences for polyploid taxa and populations occurring in isolation. Using amplified fragment length polymorphism and simple sequence repeat markers, we determined the genetic diversity and differentiation patterns at three levels of geographical isolation in Olea europaea: mainland-archipelagos, islands within an archipelago, and populations within an island. At the subspecies scale, the hexaploid ssp. maroccana (southwest Morocco) exhibited higher genetic diversity than the insular counterparts. In contrast, the tetraploid ssp. cerasiformis (Madeira) displayed values similar to those obtained for the diploid ssp. guanchica (Canary Islands). Geographical isolation was associated with a high genetic differentiation at this scale. In the Canarian archipelago, the stepping-stone model of differentiation suggested in a previous study was partially supported. Within the western lineage, an east-to-west differentiation pattern was confirmed. Conversely, the easternmost populations were more related to the mainland ssp. europaea than to the western guanchica lineage. Genetic diversity across the Canarian archipelago was significantly correlated with the date of the last volcanic activity in the area/island where each population occurs. At the island scale, this pattern was not confirmed in older islands (Tenerife and Madeira), where populations were genetically homogeneous. In contrast, founder effects resulted in low genetic diversity and marked genetic differentiation among populations of the youngest island, La Palma.

How much does the amphioxus genome represent the ancestor of chordates?

One of the main motivations to study amphioxus is its potential for understanding the last common ancestor of chordates, which notably gave rise to the vertebrates. An important feature in this respect is the slow evolutionary rate that seems to have characterized the cephalochordate lineage, making amphioxus an interesting proxy for the chordate ancestor, as well as a key lineage to include in comparative studies. Whereas slow evolution was first noticed at the phenotypic level, it has also been described at the genomic level. Here, we examine whether the amphioxus genome is indeed a good proxy for the genome of the chordate ancestor, with a focus on protein-coding genes. We investigate genome features, such as synteny, gene duplication and gene loss, and contrast the amphioxus genome with those of other deuterostomes that are used in comparative studies, such as Ciona, Oikopleura and urchin.

Nitric oxide synthase 2 is required for conversion of pro-fibrogenic inflammatory CD133+ progenitors into F4/80+ macrophages in experimental autoimmune myocarditis.

AIMS: Experimental autoimmune myocarditis (EAM) model mirrors important mechanisms of inflammatory dilated cardiomyopathy (iDCM). In EAM, inflammatory CD133(+) progenitors are a major cellular source of cardiac myofibroblasts in the post-inflammatory myocardium. We hypothesized that exogenous delivery of macrophage-colony-stimulating factor (M-CSF) can stimulate macrophage lineage differentiation of inflammatory progenitors and, therefore, prevent their naturally occurring myofibroblast fate in EAM. METHODS AND RESULTS: EAM was induced in wild-type (BALB/c) and nitric oxide synthase 2-deficient (Nos2(-/-)) mice and CD133(+) progenitors were isolated from inflamed hearts. In vitro, M-CSF converted inflammatory CD133(+) progenitors into nitric oxide-producing F4/80(+) macrophages and prevented transforming growth factor-β-mediated myofibroblast differentiation. Importantly, only a subset of heart-infiltrating CD133(+) progenitors expresses macrophage-specific antigen F4/80 in EAM. These CD133(+)/F4/80(hi) cells show impaired myofibrogenic potential compared with CD133(+)/F4/80(-) cells. M-CSF treatment of wild-type mice with EAM at the peak of disease markedly increased CD133(+)/F4/80(hi) cells in the myocardium, and CD133(+) progenitors isolated from M-CSF-treated mice failed to differentiate into myofibroblasts. In contrast, M-CSF was not effective in converting CD133(+) progenitors from inflamed hearts of Nos2(-/-) mice into macrophages, and M-CSF treatment did not result in increased CD133(+)/F4/80(hi) cell population in hearts of Nos2(-/-) mice. Accordingly, M-CSF prevented post-inflammatory fibrosis and left ventricular dysfunction in wild-type but not in Nos2(-/-) mice. CONCLUSION: Active and NOS2-dependent induction of macrophage lineage differentiation abrogates the myofibrogenic potential of heart-infiltrating CD133(+) progenitors. Modulating the in vivo differentiation fate of specific progenitors might become a novel approach for the treatment of inflammatory heart diseases.

Two alternate mechanisms contribute to the persistence of interdependent lineages in Pogonomyrmex harvester ants.

Some populations of Pogonomyrmex harvester ants comprise pairs of highly differentiated lineages with queens mating at random with several males of their own and of the alternate lineage. These queens produce two types of diploid offspring, those fertilized by males of the queens' lineage which develop into new queens and those fertilized by males of the other lineage which mostly develop into functionally sterile workers. This unusual mode of genetic caste determination has been found in 26 populations and a total of four lineage pairs (F(1)-F(2), G(1)-G(2), H(1)-H(2) and J(1)-J(2)) have been described in these populations. Despite the fact that a few interlineage queens are produced, previous studies revealed that there is a complete lack of genetic introgression between lineages. Here we quantify the proportion of interlineage queens produced in each of the four lineage pairs and determine the fate of these queens. In the F(1)-F(2), G(1)-G(2) and H(1)-H(2) lineage pairs, interlineage queens were produced by a minority of colonies. These colonies exclusively produced interlineage queens and workers, suggesting that interlineage eggs can develop into queens in these three pairs of lineages in the absence of competition with pure-lineage brood. An analysis of three key stages of the colony life cycle revealed that colonies headed by interlineage queens failed to grow sufficiently to produce reproductive individuals. In laboratory comparisons, interlineage queens produced fewer viable eggs, with the effect that they raised fewer workers and lost more weight per worker produced than pure-lineage queens. In the J(1)-J(2) lineage pair, we did not find a single interlineage queen, raising the possibility that interlineage eggs have completely lost the ability to develop into queens in this lineage pair. Hence, two distinct mechanisms seem to account for the complete lack of between-lineage gene flow in the F(1)-F(2), G(1)-G(2), H(1)-H(2) and J(1)-J(2) lineage pairs.

Differentiation of rat striatal embryonic stem cells in vitro: monolayer culture vs. three-dimensional coculture with differentiated brain cells.

Several groups have demonstrated the existence of self-renewing stem cells in embryonic and adult mouse brain. In vitro, these cells proliferate in response to epidermal growth factor, forming clusters of nestin-positive cells that may be dissociated and subcultured repetitively. Here we show that, in stem cell clusters derived from rat embryonic striatum, cell proliferation decreased with increasing number of passages and in response to elevated concentrations of potassium (30 mM KCl). In monolayer culture, the appearance of microtubule-associated protein type-5-immunoreactive (MAP-5(+)) cells (presumptive neurons) in response to basic fibroblast growth factor (bFGF) was reduced at low cell density and with increasing number of passages. In the presence of bFGF, elevated potassium caused a more differentiated neuronal phenotype, characterized by an increased proportion of MAP-5(+) cells, extensive neuritic branching, and higher specific activity of glutamic acid decarboxylase. Dissociated stem cells were able to invade cultured brain cell aggregates containing different proportions of neurons and glial cells, whereas they required the presence of a considerable proportion of glial cells in the host cultures to become neurofilament H-positive. The latter observation supports the view that astrocyte-derived factors influence early differentiation of the neuronal cell lineage.

A vertebrate reproductive system involving three ploidy levels : hybrid origin of triploids in a contact zone of diploid and tetraploid green toads (Bufo viridis subgroup)

The rise and consequences of polyploidy in vertebrates, whose origin was associated with genome duplications, may be best studied in natural diploid and polyploid populations. In a diploid/tetraploid (2n/4n) geographic contact zone of Palearctic green toads in northern Kyrgyzstan, we examine 4ns and triploids (3n) of unknown genetic composition and origins. Using mitochondrial and nuclear sequence, and nuclear microsatellite markers in 84 individuals, we show that 4n (Bufo pewzowi) are allopolyploids, with a geographically proximate 2n species (B. turanensis) being their maternal ancestor and their paternal ancestor as yet unidentified. Local 3n forms arise through hybridization. Adult 3n mature males (B. turanensis mtDNA) have 2n mothers and 4n fathers, but seem distinguishable by nuclear profiles from partly aneuploid 3n tadpoles (with B. pewzowi mtDNA). These observations suggest multiple pathways to the formation of triploids in the contact zone, involving both reciprocal origins. To explain the phenomena in the system, we favor a hypothesis where 3n males (with B. turanensis mtDNA) backcross with 4n and 2n females. Together with previous studies of a separately evolved, sexually reproducing 3n lineage, these observations reveal complex reproductive interactions among toads of different ploidy levels and multiple pathways to the evolution of polyploid lineages.

Selective abrogation of major histocompatibility complex class II expression on extrahematopoietic cells in mice lacking promoter IV of the class II transactivator gene.

MHC class II (MHCII) molecules play a pivotal role in the induction and regulation of immune responses. The transcriptional coactivator class II transactivator (CIITA) controls MHCII expression. The CIITA gene is regulated by three independent promoters (pI, pIII, pIV). We have generated pIV knockout mice. These mice exhibit selective abrogation of interferon (IFN)-gamma-induced MHCII expression on a wide variety of non-bone marrow-derived cells, including endothelia, epithelia, astrocytes, and fibroblasts. Constitutive MHCII expression on cortical thymic epithelial cells, and thus positive selection of CD4(+) T cells, is also abolished. In contrast, constitutive and inducible MHCII expression is unaffected on professional antigen-presenting cells, including B cells, dendritic cells, and IFN-gamma-activated cells of the macrophage lineage. pIV(-/-) mice have thus allowed precise definition of CIITA pIV usage in vivo. Moreover, they represent a unique animal model for studying the significance and contribution of MHCII-mediated antigen presentation by nonprofessional antigen-presenting cells in health and disease.

Absolute requirement for the pre-T cell receptor alpha chain during NK1.1+ TCRalphabeta cell development.

Most natural killer T (NKT) cells express a highly skewed alphabeta TCR repertoire, consisting of an invariant V alpha14-J alpha281 chain paired preferentially with a polyclonal Vbeta8.2 chain. This repertoire is positively selected by the monomorphic CD1d molecule expressed on cells of hematopoietic origin. The origin of NKT cells and their lineage relationship to conventional T cells is controversial. We show here that the development of NKT cells is absolutely dependent on expression of the pre-TCRalpha chain, in marked contrast to conventional T cells which arise in significant numbers even in the absence of a functional pre-TCR. Distinct developmental requirements for pre-TCR expression in the NKT and T cell lineages may reflect differences in the ability of the TCRalphabeta to substitute functionally for the pre-TCR in immature precursor cells.

Unexpectedly late expression of intracellular CD3epsilon and TCR gammadelta proteins during adult thymus development.

During adult thymus development immature CD4(-)CD8(-) [double-negative (DN)] precursor cells pass through four phenotypically distinct stages defined by expression of CD44 and CD25: CD44(hi)CD25(-) (DN1), CD44(hi)CD25(+) (DN2), CD44(lo)CD25(+) (DN3) and CD44(lo)CD25(-) (DN4). Although it is well established that the TCR beta, gamma and delta genes are rearranged and expressed in association with the CD3 components in DN thymocytes, the precise timing of expression of the TCR and CD3 proteins has not been determined. In this report we have utilized a sensitive intracellular (ic) staining technique to analyze the expression of ic CD3epsilon, TCR beta and TCR gammadelta proteins in immature DN subsets. As expected from previous studies of TCR beta rearrangement and mRNA expression, icTCR beta(+) cells were first detected in the DN3 subset and their proportion increased thereafter. Surprisingly, however, both icCD3epsilon(+) and icTCR gammadelta(+) cells were detected at later stages of development than was predicted by molecular studies. In particular icCD3epsilon protein expression coincided with the transition from the DN2 to DN3 stage of development, whereas icTCR gammadelta protein expression was only detected in a minor subset of DN4 cells. The implications of these findings for alphabeta lineage divergence will be discussed.

Transcriptional regulation of CD4 gene expression by T cell factor-1/beta-catenin pathway.

By interacting with MHC class II molecules, CD4 facilitates lineage development as well as activation of Th cells. Expression of physiological levels of CD4 requires a proximal CD4 enhancer to stimulate basic CD4 promoter activity. T cell factor (TCF)-1/beta-catenin pathway has previously been shown to regulate thymocyte survival via up-regulating antiapoptotic molecule Bcl-xL. By both loss and gain of function studies, in this study we show additional function of TCF-1/beta-catenin pathway in the regulation of CD4 expression in vivo. Mice deficient in TCF-1 displayed significantly reduced protein and mRNA levels of CD4 in CD4+ CD8+ double-positive (DP) thymocytes. A transgene encoding Bcl-2 restored survival but not CD4 levels of TCF-1(-/-) DP cells. Thus, TCF-1-regulated survival and CD4 expression are two separate events. In contrast, CD4 levels were restored on DP TCF-1(-/-) cells by transgenic expression of a wild-type TCF-1, but not a truncated TCF-1 that lacks a domain required for interacting with beta-catenin. Furthermore, forced expression of a stabilized beta-catenin, a coactivator of TCF-1, resulted in up-regulation of CD4. TCF-1 or stabilized beta-catenin greatly stimulated activity of a CD4 reporter gene driven by a basic CD4 promoter and the CD4 enhancer. However, mutation of a potential TCF binding site located within the enhancer abrogated TCF-1 and beta-catenin-mediated activation of CD4 reporter. Finally, recruitment of TCF-1 to CD4 enhancer was detected in wild-type but not TCF-1 null mice by chromatin-immunoprecipitation analysis. Thus, our results demonstrated that TCF/beta-catenin pathway enhances CD4 expression in vivo by recruiting TCF-1 to stimulate CD4 enhancer activity.

Normal hemopoiesis and lymphopoiesis in the combined absence of numb and numblike.

The mammalian ortholog of the conserved Drosophila adaptor protein Numb (Nb) and its homolog Numblike (Nbl) modulate neuronal cell fate determination at least in part by antagonizing Notch signaling. Because the Notch pathway has been implicated in regulating hemopoietic stem cell self-renewal and T cell fate specification in mammals, we investigated the role of Nb and Nbl in hemopoiesis using conditional gene targeting. Surprisingly simultaneous deletion of both Nb and Nbl in murine bone marrow precursors did not affect the ability of stem cells to self-renew or to give rise to differentiated myeloid or lymphoid progeny, even under competitive conditions in mixed chimeras. Furthermore, T cell fate specification and intrathymic T cell development were unaffected in the combined absence of Nb and Nbl. Collectively our data indicate that the Nb family of adaptor proteins is dispensable for hemopoiesis and lymphopoiesis in mice, despite their proposed role in neuronal stem cell development.

Genetic survey of "spinturnix sp" (Acari, Mesostigmata) and their bat hosts in Western Palearctic

SUMMARY : The coevolution between two intimately associated organisms, like host and parasite, is a widely investigated theme in evolutionary biology. Recently, the use of genetic data in the study of host-parasite systems evidences that the genetic information from some parasites can complement genetic data from their hosts and thus may help to better understand their host's evolutionary history. Phylogenetic and population genetic aspects of bat parasites have been poorly investigated. Spinturnicid mites are highly specialized ectoparasites, exclusively associated with bats and therefore represent an ideal model to extant our knowledge on bat and parasite biology and on their coevolutionary history. In this thesis, I developed several molecular markers (mitochondrial DNA) to compare the genetic patterns of Spinturnix mites with their bat hosts at different levels. The molecular co-phylogeny between Spinturnix sp. and their bat hosts suggests a partial cospeciation and the occurrence of failure to speciate events and multiple host switches. Thus, Spinturnix mites do not exactly mirror the phylogenetic pattern of their hosts, despite their intimate association. Similar roosting habits of the hosts seem to promote host switches between different species, as far as ecological conditions are favourable. The phylogeographic study of the Maghrebian bat M. punicus in the Mediterranean area confirms the presence of M. punicus in North Africa, Corsica and Sardinia and highlights that islands and mainland are genetically highly divergent. The comparison between the parasitic mite S. myoti and the Maghrebian bat suggests that the phylogeographic pattern of the mite is moulded by its host, with open water as main barrier for host and parasite dispersal. Moreover, the unique presence of a European S. myoti lineage on M. punicus from Corsica strongly suggests the former presence of mouse-eared bats (M. myotis and/or M. blythii) in Corsica. By highlighting the probable presence of a nowadays locally extinct host species, S. myoti may represent a good proxy for inferring complex evolutionary history of bat hosts. Finally, population genetic surveys of S. myoti and S. bechsteinii suggest that these mites benefit from close contacts between individuals during the mating season and/or hibernation to disperse among remote colonies. The contrasted genetic patterns of these two distinct bat-mite systems evidence that bat social structure is a determinant factor of the genetic structure of mite populations. Altogether, this PhD thesis demonstrates the usefulness of parasites to gather information about their bat hosts. In addition, my results illustrate how different ecological and biological characteristics of bat species allow the emergence of a surprising diversity in the genetic patterns of the parasites, which may contribute to the diversification and speciation of parasites. RESUME : La co-évolution entre deux organismes intimement liés, comme un parasite et son hôte, fait partie des questions largement étudiées en biologie évolutive. Récemment, l'utilisation de données génétique dans l'étude des interactions hôte-parasite a montré que l'information génétique de certains parasites peut compléter les données génétiques de l'hôte et ainsi peut éclairer l'histoire évolutive de leur hôte. Très peu études ont étudié les interactions entre les chauves-souris et leurs parasites d'un point de vue moléculaire. Les acariens du genre Spinturnix sont des ectoparasites très spécialisés exclusivement associés aux chauves-souris. Ils représentent donc un model idéal pour élargir nos connaissances tant sur l'écologie des parasites de chauves-souris que sur leur coévolution. Durant cette thèse, plusieurs marqueurs moléculaires (ADN mitochondrial) ont été développés pour ainsi comparer la distribution de la variation génétique des parasites du genre Spinturnix avec celle de leurs hôtes, et ceci à différents niveaux. Tout d'abord, la co-phylogénie moléculaire entre les espèces de Spinturnix et les leurs hôtes révèle une co-spéciation partielle ainsi que la présence d'événement de non spéciation et de transferts horizontaux. Ces parasites ne reflètent donc pas entièrement l'histoire évolutive de leurs hôtes, malgré leurs intimes associations. La cohabitation de plusieurs espèces de chauves-souris dans un même gîte permet aux parasites un transfert entre différentes espèces, atténuant ainsi leur degré de co-spéciation. Deuxièmement, l'étude phylogéographique du marin du Maghreb dans le bassin Méditerranéen confirme sa présence en Afrique du Nord, en Corse et en Sardaigne. La comparaison avec un de ses parasites S. myoti suggère que la répartition génétique de S. myoti est façonnée par celle de leurs hôtes, avec les étendues d'eau comme barrière principale tant à la dispersion de l'hôte que de son parasite. De plus, la présence unique d'une lignée européenne de ces parasites sur des marins du Maghreb de Corse suggère fortement la présence du grand ou petit marin en Corse dans le passé. En reflétant la présence potentielle à un endroit donné d'une espèce de chauve-souris actuellement disparue, S. myoti peut représenter une bonne alternative pour comprendre l'histoire évolutive complexe des chauves-souris. Finalement, l'étude des structures génétiques des populations des parasites S. myoti et S. bechsteinii suggère que les contacts corporels entre chauves-souris durant la saison de reproduction ou l'hibernation peuvent permettre la dispersion des parasites entre des colonies éloignées géographiquement. La différence de structure génétique entre ces deux associations particulières montre que la structure génétique des populations de parasites dépend fortement des traits d'histoire de vie de son hôte. Dans l'ensemble, cette thèse démontre l'importance des parasites pour amener des informations sur leurs hôtes, les chauves-souris. Elle illustre aussi comment les différences écologique et biologique des différentes espèces de chauves-souris peuvent amener une étonnante diversité de structure génétique au sein de populations de parasites, ce qui peut peut-être contribuer à la diversification et à la spéciation des parasites.

Development and selection of NKT cells.

NKT cells utilize a restricted alphabeta TCR repertoire that recognizes glycolipids in association with CD1d. The recent development of fluorescent CD1d tetramers loaded with the synthetic glycolipid alpha-galactosyl-ceramide has led to a clearer definition of NKT-cell subsets as well as important insights into their developmental origin. As many as four subsets may exist, differing in NK1.1 expression, TCR repertoire and dependence on CD1d and various glycolipids for development. Two different lineage-commitment models have been proposed, with most evidence favoring a byproduct of conventional-T-cell development.

Estrogen receptor regulates MyoD gene expression by preventing AP-1-mediated repression.

Cell growth and differentiation are opposite events in the myogenic lineage. Growth factors block the muscle differentiation program by inducing the expression of transcription factors that negatively regulate the expression of muscle regulatory genes like MyoD. In contrast, extracellular clues that induce cell cycle arrest promote MyoD expression and muscle differentiation. Thus, the regulation of MyoD expression is critical for muscle differentiation. Here we show that estrogen induces MyoD expression in mouse skeletal muscle in vivo and in dividing myoblasts in vitro by relieving the MyoD promoter from AP-1 negative regulation through a mechanism involving estrogen receptor/AP-1 protein-protein interactions but independent of the estrogen receptor DNA binding activity.