Calibration of the low-energy channel Thomson parabola of the LMJ-PETAL diagnostic SEPAGE with protons and carbon ions.

The SEPAGE diagnostic will detect charged particles (electrons, protons, and ions) accelerated in the interaction of the PETAL (PETawatt Aquitaine Laser) laser with its targets on the LMJ (Laser MegaJoule)-PETAL laser facility. SEPAGE will be equipped with a proton-radiography front detector and two Thomson parabolas (TP), corresponding to different ranges of the particle energy spectra: Above 0.1 MeV for electrons and protons in the low-energy channel, with a separation capability between protons and 12C6+ up to 20 MeV proton energy and above 8 MeV for the high-energy channel, with a separation capability between protons and 12C6+ up to 200 MeV proton kinetic energy. This paper presents the calibration of the SEPAGE's low-energy channel TP at the Tandem facility of Orsay (France) with proton beams between 3 and 22 MeV and carbon-ion beams from 5.8 to 84 MeV. The magnetic and electric fields' integrals were determined with an accuracy of 10-3 by combining the deflections measured at different energies with different target thicknesses and materials, providing different in-target energy losses of the beam particles and hence different detected energies for given beam energies.

Calibration of imaging plate detectors to mono-energetic protons in the range 1-200 MeV.

Responses of Fuji Imaging Plates (IPs) to proton have been measured in the range 1-200 MeV. Mono-energetic protons were produced with the 15 MV ALTO-Tandem accelerator of the Institute of Nuclear Physics (Orsay, France) and, at higher energies, with the 200-MeV isochronous cyclotron of the Institut Curie-Centre de Protonthérapie d'Orsay (Orsay, France). The experimental setups are described and the measured photo-stimulated luminescence responses for MS, SR, and TR IPs are presented and compared to existing data. For the interpretation of the results, a sensitivity model based on the Monte Carlo GEANT4 code has been developed. It enables the calculation of the response functions in a large energy range, from 0.1 to 200 MeV. Finally, we show that our model reproduces accurately the response of more complex detectors, i.e., stack of high-Z filters and IPs, which could be of great interest for diagnostics of Petawatt laser accelerated particles.

Direct observation of two protons in the decay of 54Zn.

The two protons emitted in the decay of 54Zn have been individually observed for the first time in a time projection chamber. The total decay energy and the half-life measured in this work agree with the results obtained in a previous experiment. Angular and energy correlations between the two protons are determined and compared to theoretical distributions of a three-body model. Within the shell model framework, the relative decay probabilities show a strong contribution of the p2 configuration for the two-proton emission. After 45Fe, the present result on 54Zn constitutes only the second case of a direct observation of the ground state two-proton decay of a long-lived isotope.

Regeneration of dendritic cells in aged mice.

Age-related thymic involution causes a decreased output of thymocytes from the thymus, thereby resulting in impairment of T cell-mediated immunity. While alterations in the T cell and non-haematopoietic stromal compartments have been described, the effects of thymic involution on thymic dendritic cells (DC) are not clearly known. Thymic DC play an essential role in shaping T cell-mediated immune responses by deleting self-reactive thymocytes to establish central tolerance and by inducing regulatory T-cell (Treg) development. It is therefore important to assess the prevalence of and alterations to thymic DC with age, as this may impact on their function. We assessed the numbers and proportions of the three distinct subsets of thymic DC in ageing mice, and showed that these subsets are differentially regulated. This is expected as thymic DC subsets have different origins of development. We further assessed the responses of thymic DC in a regenerative environment, such as that induced by sex-steroid ablation (SSA), and clearly showed that, consistent with global thymus regrowth, all three DC populations increased in numbers and regained their relative proportions to thymocytes after an initial lag period. These findings are important for the clinical translation of thymic regenerative approaches, and indicate that SSA facilitates the maintenance of critical processes such as negative selection and Treg induction through promoting thymic DC regeneration.

The roles of interferon-gamma and perforin in antiviral immunity in mice that differ in genetically determined NK-cell-mediated antiviral activity.

The design of effective antiviral immunotherapies depends on a detailed understanding of the cellular and molecular processes involved in generating and maintaining immune responses. Control of cytomegalovirus (CMV) infection requires the concerted activities of both innate and adaptive immune effectors. In the mouse, immunity to acute murine CMV (MCMV) infection depends on natural killer (NK) cells and/or CD8(+) T cells. The relative importance of NK and CD8(+) T cells varies in different mouse strains. In C57BL/6 mice, early viral infection is controlled by Ly49H(+) NK cells, whereas in BALB/c mice, CD8(+) T cells exert the principal antiviral activities. Although the role of NK and CD8(+) T cells is defined, the molecular mechanisms they utilize to limit acute infection are poorly understood. Here, we define the specific roles of perforin (pfp) and interferon-gamma (IFN-gamma) in the context of NK- or T-cell-mediated immunity to MCMV during acute infection. We show that pfp is essential for both NK- and T-cell-mediated antiviral immunity during the early stages of infection. The relative importance of IFN-gamma is more pronounced in Ly49H(-) mice. Using BALB/c background mice congenic for Ly49H and lacking pfp, we show that Ly49H-regulated NK-cell control of MCMV infection is dependent on pfp-mediated cytolysis.

The dendritic cell subtype-restricted C-type lectin Clec9A is a target for vaccine enhancement.

A novel dendritic cell (DC)-restricted molecule, Clec9A, was identified by gene expression profiling of mouse DC subtypes. Based on sequence similarity, a human ortholog was identified. Clec9A encodes a type II membrane protein with a single extracellular C-type lectin domain. Both the mouse Clec9A and human CLEC9A were cloned and expressed, and monoclonal antibodies (mAbs) against each were generated. Surface staining revealed that Clec9A was selective for mouse DCs and was restricted to the CD8(+) conventional DC and plasmacytoid DC subtypes. A subset of human blood DCs also expressed CLEC9A. A single injection of mice with a mAb against Clec9A, which targets antigens (Ags) to the DCs, produced a striking enhancement of antibody responses in the absence of added adjuvants or danger signals, even in mice lacking Toll-like receptor signaling pathways. Such targeting also enhanced CD4 and CD8 T-cell responses. Thus, Clec9A serves as a new marker to distinguish subtypes of both mouse and human DCs. Furthermore, targeting Ags to DCs with antibodies to Clec9A is a promising strategy to enhance the efficiency of vaccines, even in the absence of adjuvants.

The radioactive ion beam production systems for the SPIRAL2 project.

The SPIRAL2 project, currently under construction at GANIL, will include an isotope separator on line based facility for the production and acceleration of radioactive ion beams. A superconducting linear accelerator will accelerate 5 mA deuterons up to 40 MeV and 1 mA heavy ions up to 14.5 MeV/u. These primary beams will be used to bombard both thick and thin targets. We are investigating three different techniques to produce the radioactive ion beams: (1) the neutron induced fission of uranium carbide, (2) the direct interaction of deuterons in a uranium carbide target, and (3) the interaction of a heavy ion beam with a target. All these production systems will be coupled to an ion source. Four kinds of ion sources are foreseen for the ionization of the radioactive atoms: an electron cyclotron resonance ion source, a surface ionization ion source, a forced electron beam induced arc discharge ion source, and a laser ion source depending on the characteristics of the desired radioactive ion beam in terms of intensity, efficiency, purity, etc. A presentation of the SPIRAL2 project and of the different production systems is given.

First direct observation of two protons in the decay of 45Fe with a time-projection chamber.

The decay of the ground-state two-proton emitter 45Fe was studied with a time-projection chamber and the emission of two protons was unambiguously identified. The total decay energy and the half-life measured in this work agree with the results from previous experiments. The present result constitutes the first direct observation of the individual protons in the two-proton decay of a long-lived ground-state emitter. In parallel, we identified for the first time directly two-proton emission from 43Cr, a known beta-delayed two-proton emitter. The technique developed in the present work opens the way to a detailed study of the mechanism of ground state as well as beta-delayed two-proton radioactivity.

Perforin and granzymes have distinct roles in defensive immunity and immunopathology.

Successful control of viral infection requires the host to eliminate the infecting pathogen without causing overt immunopathology. Here we showed that perforin (Prf1) and granzymes (Gzms) have distinct roles in defensive immunity and immunopathology in a well-established model of viral infection. Both Prf1 and Gzms drastically affected the outcome of murine cytomegalovirus (MCMV) infection. Viral titres increased markedly in both Prf1(-/-) and Gzma(-/-)Gzmb(-/-) mice, but Gzma(-/-)Gzmb(-/-) mice recovered and survived infection, whereas Prf1(-/-) mice did not. Indeed, infected Prf1-deficient hosts developed a fatal hemophagocytic lymphohistiocytosis (HLH)-like syndrome. This distinction in outcome depended on accumulation of mononuclear cells and T cells in infected Prf1(-/-) mice. Importantly, blocking experiments that clearly identified tumor necrosis factor-alpha (TNF-alpha) as the principal contributor to the lethality observed in infected Prf1(-/-) mice provided support for the clinical potential of such an approach in HLH patients whose disease is triggered by viral infection.

Functional comparison of mouse CIRE/mouse DC-SIGN and human DC-SIGN.

CIRE/mDC-SIGN is a C-type lectin we originally identified as a molecule differentially expressed by mouse dendritic cell (DC) populations. Immunostaining with a CIRE/mDC-SIGN-specific mAb revealed that CIRE/mDC-SIGN is indeed on the surface of some CD4+, CD4- 8- DCs and plasmacytoid pre-DCs, but not on CD8+ DCs. It has been proposed that CIRE/mDC-SIGN is the functional orthologue of human DC-SIGN (hDC-SIGN), a molecule that both enhances T cell responses and facilitates antigen uptake. We assessed if CIRE/mDC-SIGN and hDC-SIGN exhibit functional similarities. CIRE/mDC-SIGN is down-regulated upon activation, but unlike hDC-SIGN, incubation with IL-4 and IL-13 did not enhance CIRE/mDC-SIGN expression, indicating differences in gene regulation. Like hDC-SIGN, CIRE/mDC-SIGN bound mannosylated residues. However, we could detect no role for CIRE/mDC-SIGN in T cell-DC interactions and the protein did not bind to pathogens known to interact with hDC-SIGN, including Leishmania mexicana, cytomegalovirus, HIV and lentiviral particles bearing the Ebolavirus glycoprotein. The binding of CIRE/mDC-SIGN to hDC-SIGN ligands was not rescued when CIRE/mDC-SIGN was engineered to express the stalk region of hDC-SIGN. We conclude that there are significant differences in the fine specificity of the C-type lectin domains of hDC-SIGN and CIRE/mDC-SIGN and that these two molecules may not be functional orthologues.

Interaction between conventional dendritic cells and natural killer cells is integral to the activation of effective antiviral immunity.

Dendritic cells (DCs) regulate various aspects of innate immunity, including natural killer (NK) cell function. Here we define the mechanisms involved in DC-NK cell interactions during viral infection. NK cells were efficiently activated by murine cytomegalovirus (MCMV)-infected CD11b(+) DCs. NK cell cytotoxicity required interferon-alpha and interactions between the NKG2D activating receptor and NKG2D ligand, whereas the production of interferon-gamma by NK cells relied mainly on DC-derived interleukin 18. Although Toll-like receptor 9 contributes to antiviral immunity, we found that signaling pathways independent of Toll-like receptor 9 were important in generating immune responses to MCMV, including the production of interferon-alpha and the induction of NK cell cytotoxicity. Notably, adoptive transfer of MCMV-activated CD11b(+) DCs resulted in improved control of MCMV infection, indicating that these cells participate in controlling viral replication in vivo.

Activation of NK cell cytotoxicity.

Natural killer (NK) cells are innate effector lymphocytes necessary for defence against stressed, microbe-infected, or malignant cells. NK cells kill target cells by either of two major mechanisms that require direct contact between NK cells and target cells. In the first pathway, cytoplasmic granule toxins, predominantly a membrane-disrupting protein known as perforin, and a family of structurally related serine proteases (granzymes) with various substrate specificities, are secreted by exocytosis and together induce apoptosis of the target cell. The granule-exocytosis pathway potently activates cell-death mechanisms that operate through the activation of apoptotic cysteine proteases (caspases), but can also cause cell death in the absence of activated caspases. The second pathway involves the engagement of death receptors (e.g. Fas/CD95) on target cells by their cognate ligands (e.g. FasL) on NK cells, resulting in classical caspase-dependent apoptosis. The comparative role of these pathways in the pathophysiology of many diseases is being dissected by analyses of gene-targeted mice that lack these molecules, and humans who have genetic mutations affecting these pathways. We are also now learning that the effector function of NK cells is controlled by interactions involving specific NK cell receptors and their cognate ligands, either on target cells, or other cells of the immune system. This review will discuss the functional importance of NK cell cytotoxicity and the receptor/ligand interactions that control these processes.

Cross-talk between dendritic cells and natural killer cells in viral infection.

Dendritic cells (DC), first characterized in 1973 by Steinman and Cohn, have been defined as the professional antigen presenting cells (APC), capable of activating naïve T cells much more efficiently than either B cells or macrophages. DC also capture and process antigen more efficiently than other APC, and offer MHC-antigen complexes to T cells at higher densities, and in the context of larger amounts of co-stimulatory molecules (i.e. CD40, CD80 and CD86) at the T cell-DC synapse. Although historically, the principal function of DC is the priming of naïve T cells, more recently they have also been shown to affect the functions of natural killer (NK) cells. Interactions between DC and NK cells may be critical in situations where immune surveillance requires efficient early activation of NK cells, as is the case during infections. This review aims to summarise the interactions that occur between DC and NK cells during viral infection.

A contribution of mouse dendritic cell-derived IL-2 for NK cell activation.

Dendritic cells (DCs) play a predominant role in activation of natural killer (NK) cells that exert their functions against pathogen-infected and tumor cells. Here, we used a murine model to investigate the molecular mechanisms responsible for this process. Two soluble molecules produced by bacterially activated myeloid DCs are required for optimal priming of NK cells. Type I interferons (IFNs) promote the cytotoxic functions of NK cells. IL-2 is necessary both in vitro and in vivo for the efficient production of IFNgamma, which has an important antimetastatic and antibacterial function. These findings provide new information about the mechanisms that mediate DC-NK cell interactions and define a novel and fundamental role for IL-2 in innate immunity.

NKT cells and viral immunity.

Over the past 10 years a new population of cells has been the focus of much attention. The functions of these unique lymphocytes, characterized by the concomitant expression of T- and NK-cell markers and thus termed NKT cells, have been implicated in many diverse aspects of immunity, including regulation of autoimmune disorders, control of tumour growth and spread, and defence against a number of pathogens. Although much debate still remains as to the natural role of NKT cells, it is clear that these cells have the capacity, either constitutively or postactivation, to promote an amazing array of immunoregulatory responses. The involvement of NKT cells in viral immune-surveillance and their ability to induce protection against pathogens once activated make them an attractive clinical target.

Activation of natural killer (NK) T cells during murine cytomegalovirus infection enhances the antiviral response mediated by NK cells.

NK1.1+ T (NKT) cells are efficient regulators of early host responses which have been shown to play a role in tumor surveillance. The relevance of NKT cells in immune surveillance of viral infections, however, is not well understood. In this study, we investigated the functional relevance of NKT cells in controlling herpesvirus infections by using challenge with murine cytomegalovirus (MCMV) as the study model. This model has proven to be one of the best systems for evaluating the role of NK cells during virus infection. Using gene-targeted mice and alpha-galactosylceramide (alpha-GalCer) as an exogenous stimulator of NKT cells, we have analyzed the role of these cells in the immune surveillance of MCMV infection. Our studies in NKT-cell-deficient, T-cell receptor Jalpha281 gene-targeted mice have established that classical NKT cells do not play a critical role in the early clearance of MCMV infection. Importantly, however, activation of NKT cells by alpha-GalCer resulted in reduced viral replication in visceral organs. Depletion studies, coupled with analysis of gene-targeted mice lacking perforin and gamma interferon (IFN-gamma), have revealed that the antiviral effects of alpha-GalCer involve NK cells and have clearly demonstrated that the antiviral activity of alpha-GalCer, unlike the antitumor one, is critically dependent on both perforin and IFN-gamma.

Sesquiterpenes and alkaloids from Scorodocarpus borneensis.

A new sesquiterpene, scodopin, and a mixture of three tryptamine-type alkaloids, scorodocarpines A-C, were isolated from the fruits of Scorodocarpus borneensis, together with a known hemisynthetic sesquiterpene, cadalene-beta-carboxylic acid, which was isolated from the bark. The structures of the new compounds were elucidated by interpretation of spectral data, especially tandem mass spectrometry for the alkaloid mixture.

Trypanocidal withanolides and withanolide glycosides from Dunalia brachyacantha.

Two new withanolide glycosides, (20R,22R)-O-(3)-alpha-L-rhamnopyranosyl(1-->4)-beta-D-glucopyranosyl-1 alpha,12 beta-diacetoxy-20-hydroxywitha-5,24-dienolide (3) and (20R,22R)-O-(3)-beta-D-xylopyranosyl(1-->3)-[beta-D-xylopyranosyl(1-->4)]-beta-D-glucopyranosyl-1 alpha-acetoxy-12 beta,20-dihydroxywitha-5,24-dienolide (4), were isolated from the leaves and root of Dunalia brachyacantha. Their aglycones, (20R,22R)-1 alpha,12 beta-diacetoxy-3 beta,20-dihydroxywitha-5,24-dienolide (or 1 alpha,12 beta-diacetyldunawithagenine) and (20R,22R)-1 alpha-acetoxy-3 beta,12 beta,20-trihydroxywitha-5,24-dienolide (or 1 alpha-acetyl-12 beta-hydroxydunawithagenine), are novel. The known 18-acetoxywithanolide D (1) and 18-acetoxy-5,6-deoxy-5-withenolide D (2) were also isolated from the leaves. These last two compounds were shown to be responsible for the trypanocidal, leishmanicidal, and bactericidal activities manifested by the crude ethanolic extract. The structures were deduced from spectroscopic data and on the basis of chemical evidence.

Stabilization of gas-phase noncovalent macromolecular complexes in electrospray mass spectrometry using aqueous triethylammonium bicarbonate buffer.

The use of triethylammonium bicarbonate (TEAB) solution in electrospray mass spectrometry proved to be a very efficient way for studying proteins or noncovalent protein complexes under "nondenaturing" conditions. The low charge states observed in the mass spectra improve the separation of ions arising from macromolecular species of close masses. Moreover, the multiply charged ions generated in a TEAB solution are significantly more stable than those formed under more conventional conditions (for example, with ammonium bicarbonate or acetate solution). The analytical interest of TEAB for the analysis of macromolecular species that can easily dissociate in the gas phase, such as hemoglobin or other macromolecular noncovalent complexes, is demonstrated.

Differential chemical labeling of the AlcR DNA-binding domain from Aspergillas nidulans versus its complex with a 16-mer DNA target: identification of an essential tryptophan involved in the recognition and the interaction with the nucleic acid.

DNA binding of the ethanol regulon transcription factor AlcR from Aspergillus nidulans was shown to involve a consensus basic region as in the other zinc cluster proteins. However, additional interactions between some residues and DNA were suspected, among which were a hypothetic hydrophobic interaction between Trp45 and the T residue of the consensus TGCGG sequence. In the present study, the differential chemical labeling of both the free protein and the protein/DNA complex showed significantly different behaviors of the three tryptophan residues comprised in the AlcR sequence toward the Koshland reagent. The spectacular decreased reaction rate for Trp45 within the complex confirmed the location of this residue at the protein/DNA interface. A similar result obtained with Trp53, an amino acid present at the C-terminal side of AlcR, also indicated its involvement in the DNA recognition. In contrast, the formation of the complex accompanied by an allosteric rearrangement allowed the Trp36 to be much more exposed to the solvent than in the free protein. These data provide additional evidence that the unique specificity of AlcR among the zinc binuclear cluster family results in new types of interactions between AlcR and its cognate targets. From a methodological point of view, the approach of differential chemical labeling combined with mass spectrometric analyses proved to be an interesting tool for the recognition of hydrophobic interactions between the tryptophan residues of a protein and its macromolecular target.