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IntroductionHematopoiesis is maintained by a pool of hematopoietic stem cells (HSCs) defined by their capacity to self-renew and, hence, to maintain the pool and to differentiate into all mature progenies. These properties underlie their ability to reconstitute the hematopoietic system. 1 HSCs are identified by molecular markers 2 and are divided into long-term HSCs (LT-HSCs), short-term HSCs (STHSCs), and multilineage progenitors (MPPs). 3,4 As they differentiate, HSCs give rise to committed progenitors, such as common lymphoid progenitors (CLPs) or common myeloid progenitors (CMPs), which generate mature cells within the different hematopoietic lineages. 5 The action of HSCs to self-renew or to differentiate is a tightly controlled process linked to the induction or repression of some crucial genes. 6 Gene-targeting experiments have provided support to this hypothesis by identifying transcription factors of various families or their coregulators that are implicated in HSC biology. 7,8 The basic helix-loop-helix (bHLH) family of transcription factors regulates a wide range of developmental events. 1,9 Some bHLHs, such as MyoD and NeuroD, display restricted tissue expression and control the differentiation of particular cellular lineages. 10 They form transactivating dimers by heteromerization with ubiquitous bHLH proteins, termed E proteins, such as E2A, E12, and E47 genes. 11,12 Conversely, the latter proteins are repressed by other HLH proteins, which often precludes differentiation. 10,13,14 The tal/scl gene (hereafter referred to as scl) encodes a bHLH protein. 15,16 It was identified through its implication in T-cell acute lymphocytic leukemia (T-ALL). Knock-out (KO) experiments 17,18 show that scl is autonomously required for primitive and definitive hematopoiesis. 19,20 Moreover, its enforced expression enhances megakaryocytopoiesis and erythropoiesis. 21,22 The Lyl-1 gene encodes a bHLH protein closely related to scl. 23 Its transcriptional activation upon translocation is also associated with T-ALL. 24 SCL and LYL-1 bHLH regions show 82% of amino acid identity, 24 suggesting that these 2 proteins share at least some target genes and biologic functions. 25,26 However, LYL-1 and SCL diverge largely outside the bHLH region and display a distinct expression pattern in hematopoietic cells. 23,27 From the Insitut National de la The biologic functions of SCL prompted us to investigate those of LYL-1 in hematopoiesis. In contrast to scl Ϫ/Ϫ mice, Lyl-1 Ϫ/Ϫ mice do not exhibit embryonic lethality but have a reduced number of B cells. Although the CLP compartment is normal, the immature B-cell compartments are reduced in adult mice. In addition, we show that Lyl-1 is highly expressed in stem/progenitor cells, correlating with an important role in the control of the size and function of this cell compartment. Similarly, the number of multipotent progenitor S 12 colony-forming units (CFU-S 12 s) is reduced in Lyl-1 Ϫ/Ϫ animals. Overall, these defects are distinct from those revealed upon scl KO, suggesting that...

Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The micronemal protein MIC3, which is a potent adhesin of T. gondii, could be a significant candidate vaccine against toxoplasmosis. In this study, all CBA/J mice intramuscularly vaccinated with a plasmid encoding the immature form of the MIC3 protein (pMIC3i) produced specific anti-MIC3 immunoglobulin G (IgG) antibodies, and their sera displayed high antibody titers. This response was increased by the coadministration of a plasmid encoding the granulocyte-macrophage colony-stimulating factor (pGM-CSF). Similarly, a specific and significant cellular immune response was obtained in mice immunized with pMIC3i, and this response was markedly enhanced by pGM-CSF coadministration. The cellular immune response was associated with the production of gamma interferon IFN-␥ and interleukin-2 (IL-2), indicating that this was a Th1-type response. This was confirmed by the production of large amounts of IgG2a. Mice immunized with pMIC3i displayed significant protection against an oral challenge with T. gondii 76K cysts, exhibiting fewer brain cysts than did the control mice. Coadministration of pGM-CSF enhanced this protection. In conclusion, this study describes the design of a potent DNA vaccine encoding the novel T. gondii target antigen, MIC3 protein, that elicits a strong specific immune response as well as providing effective protection against T. gondii infection. In the attempt to achieve complete protection against toxoplasmosis, MIC3 is a good candidate vaccine which could be combined with other relevant and previously described candidates, such as SAG1 and GRA4.

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In the human lymphoblastoid T cell line JJhan-5.1, stably transfected with a human immunodeficiency virus-1 long terminal repeat luciferase vector, the level of luciferase activity is dependent on activation of the nuclear factor B (NF-B) transcription factor. Tumor necrosis factor-induced luciferase activity was not modified in JJhan-5.1 cells co-cultivated with murine adenocarcinoma EMT-6 cells but was strongly decreased when nitric oxide (NO) synthase 2 expression was induced in these cells. Two NO synthase inhibitors counteracted this inhibitory effect. Tumor necrosis factor-␣ binding to JJhan-5.1 cells was not modified after incubation with EMT-6 cells. Viability and protein synthesis in JJhan-5.1 cells were also unchanged. Induction of NF-B DNA binding activity was inhibited when EMT-6 cells expressed NO synthase 2 activity. Aminoguanidine, which completely abolished nitrite production, prevented this inhibition. NF-B activation was also strongly inhibited by S-nitrosoglutathione but was marginally affected by N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylenediamine. Taken together, these results indicated that NO-related species, released by EMT-6 effector cells and probably different from NO itself, inhibited NF-B activation in human lymphoblastoid target cells. Consequently, transcriptional activity of a long terminal repeat-driven luciferase gene construct was markedly diminished.

In vitro selection was performed to identify DNA aptamers against the TAR RNA stem-loop structure of HIV-1. A counterselection step allowed the elimination of kissing complex-forming aptamers previously selected (Boiziau et al. J. Biol. Chem. 1999; 274:12730). This led to the emergence of oligonucleotides, most of which contained two consensus sequences, one targeted to the stem 3'-strand (5'-CCCTAGTTA) and the other complementary to the TAR apical loop (5'-CTCCC). The best aptamer could be shortened to a 19-mer oligonucleotide, characterized by a dissociation constant of 50 nM. A 16-mer oligonucleotide complementary to the TAR stem 3'-strand could also be derived from the identified aptamers, with an equal affinity (Kd = 50 nM). Experiments performed to elucidate the interaction between TAR and the aptamers (UV melting measures, enzymatic and chemical footprints) demonstrated that the TAR stem 5'-strand was not simply displaced as a result of the complex formation but unexpectedly remained associated on contact with the antisense oligonucleotide. We suggest that a multistranded structure could be formed.

In vitro selection with either DNA or RNA libraries was performed against the TAR RNA element of HIV-1. The role of the selection conditions on the outcome of the selection was evaluated by varying the magnesium concentration and the temperature. The selection stringency was demonstrated to determine i) the affinity of the best identified aptamers for the TAR target, and ii) the type of interaction between the two partners. Selections performed with a DNA library under low (4 degrees C, 10 mM magnesium) and high stringency (23 degrees C, 3 mM magnesium) led to the emergence of "kissing aptamers"; but even if the motif interacting directly with the TAR loop were identical in the two kinds of aptamers, the consensus was extended from eight to thirteen nucleotides when the Mg(2+) concentration was decreased from 10 to 3 mM. Similar kissing aptamers were selected at 23 degrees C and 37 degrees C starting with two different RNA libraries under identical ionic conditions. In addition, selection performed at 37 degrees C yielded a significant proportion of antisense sequences. Only antisense RNAs complementary to the TAR loop competitively inhibited the association of a Tat peptide with TAR.