Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration.

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

A quantitative method for measuring phototoxicity of a live cell imaging microscope.

Fluorescence-based imaging regimes require exposure of living samples under study to high intensities of focused incident illumination. An often underestimated, overlooked, or simply ignored fact in the design of any experimental imaging protocol is that exposure of the specimen to these excitation light sources must itself always be considered a potential source of phototoxicity. This can be problematic, not just in terms of cell viability, but much more worrisome in its more subtle manifestation where phototoxicity causes anomalous behaviors that risk to be interpreted as significant, whereas they are mere artifacts. This is especially true in the case of microbial pathogenesis, where host-pathogen interactions can prove especially fragile to light exposure in a manner that can obscure the very processes we are trying to observe. For these reasons, it is important to be able to bring the parameter of phototoxicity into the equation that brings us to choose one fluorescent imaging modality, or setup, over another. Further, we need to be able to assess the risk that phototoxicity may occur during any specific imaging experiment. To achieve this, we describe here a methodological approach that allows meaningful measurement, and therefore relative comparison of phototoxicity, in most any variety of different imaging microscopes. In short, we propose a quantitative approach that uses microorganisms themselves to reveal the range over which any given fluorescent imaging microscope will yield valid results, providing a metrology of phototoxic damage, distinct from photobleaching, where a clear threshold for phototoxicity is identified. Our method is widely applicable and we show that it can be adapted to other paradigms, including mammalian cell models.

Cholinergic differentiation of neural progenitors in adult mouse motor facial nucleus.

Environmental cues are critical determinants of the fate of neural progenitors (NPs) upon transplantation into the central nervous system. In the present study, we assessed the differentiation potential of NPs implanted in a cholinergic environment of the adult mouse brain. Neurospheres containing NPs issued from fetal ganglionic eminences of transgenic mice expressing the green fluorescent protein (GFP) were transplanted either inside or outside the mouse cholinergic facial motor nucleus. In some mice, a pre-degenerated nerve releasing trophic factors was grafted into this nucleus to favor NP survival and improve axonal growth into the graft. The fate of NPs was analyzed 6 to 9 days or 2 months post-transplantation by immunofluorescence under confocal microscopy. Transplanted NPs were observed both inside and outside the facial nucleus after 6 to 9 days, but almost exclusively inside after 2 months regardless of the presence of a pre-degenerated nerve. NPs expressed markers of undifferentiated cells, astrocytes, oligodendrocytes, neurons, or cholinergic cells. The cholinergic phenotype of NPs engrafted inside the facial nucleus increased with time and the presence of a pre-degenerated nerve. Large GFP cholinergic somata and abundant long cholinergic GFP axons projecting into the nerve graft were also observed. Our results show that NPs, isolated from fetal mouse brain and transplanted into the non-neurogenic environment of the adult mouse facial nucleus, differentiate into cholinergic cells capable to project axons. This environment and the nerve graft favored NP differentiation into cholinergic neurons.

Live imaging of emerging hematopoietic stem cells and early thymus colonization.

We recently demonstrated in zebrafish the developmental migration of emerging hematopoietic stem cells (HSCs) that is thought to occur in mammalian embryos, from the aorta-gonad-mesonephros (AGM) area to the successive hematopoietic organs. CD41 is the earliest known molecular marker of nascent HSCs in mammalian development. In this study, we show that in CD41-green fluorescent protein (GFP) transgenic zebrafish embryos, the transgene is expressed by emerging HSCs in the AGM, allowing us for the first time to image their behavior and trace them in real time. We find that the zebrafish AGM contains no intra-aortic cell clusters, so far considered a hallmark of HSC emergence. CD41GFP(low) HSCs emerge in the subaortic mesenchyme and enter the circulation not through the dorsal aorta but through the axial vein, the peculiar structure of which facilitates their intravasation. The rise in CD41-gfp expression among c-myb(+) HSC precursors is asynchronous and marks their competence to leave the AGM and immediately seed the caudal hematopoietic tissue (which has a hematopoietic function analogous to that of the mammalian fetal liver). Imaging the later migration of CD41-GFP(+) precursors to the nascent thymus reveals that although some reach the thymus by extravasating from the nearest vein, most travel for hours through the mesenchyme from surprisingly diverse and remote sites of extravasation.

An extracellular matrix glues together the aerial-grown hyphae of Aspergillus fumigatus.

Pulmonary infections due to Aspergillus fumigatus result from the development of a colony of tightly associated hyphae in contact with the air, either in the alveoli (invasive aspergillosis) or in an existing cavity (aspergilloma). The fungal ball observed in vivo resembles an aerial colony obtained in agar medium in vitro more than a mycelial mass obtained in liquid shaken conditions that have been classically used to date to study A. fumigatus physiology. For this reason, we embarked on an analysis of the characteristics of A. fumigatus colonies grown in aerial static conditions. (i) Under static aerial conditions, mycelial growth is greater than in shaken, submerged conditions. (ii) The colony surface of A. fumigatus revealed the presence of an extracellular hydrophobic matrix that acts as a cohesive linkage bonding hyphae into a contiguous sheath. (iii) The extracellular matrix is composed of galactomannan, alpha1,3 glucans, monosaccharides and polyols, melanin and proteins including major antigens and hydrophobins. (iv) A. fumigatus colonies were more resistant to polyenes than shake, submerged mycelium. This is the first analysis of the three dimensional structure of a mycelial colony. Knowledge of this multicellular organization will impact our future understanding of the pathobiology of aerial mold pathogens.

Quantitative four-dimensional tracking of cytoplasmic and nuclear HIV-1 complexes.

Emerging real-time techniques for imaging viral infections provide powerful tools for understanding the dynamics of virus-host cell interactions. Here we labeled human immunodeficiency virus-1 (HIV-1) integrase with a small tetracysteine tag, which preserved the virus' infectivity while allowing it to be labeled with the bis-arsenical fluorescein derivative FlAsH. This labeling allowed us to image both intracytoplasmic and intranuclear HIV-1 complexes in three dimensions over time (4D) in human cells and enabled us to analyze HIV-1 kinetics by automated 4D quantitative particle tracking. In the cytoplasm, HIV-1 complexes underwent directed movements toward the nuclear compartment, kinetically characteristic of both microtubule- and actin-dependent transport. The complexes then adopted smaller movements in a very confined volume once associated with the nuclear membrane and more diffuse movements once inside the nucleus. This work contributes new insight into the various movements of HIV-1 complexes within infected cells and provides a useful tool for the study of virus-host cell interactions during infection.

Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin.

Mycobacterium ulcerans (Mu), the aetiological agent of Buruli ulcer, is an extracellular pathogen producing the macrolide toxin mycolactone. Using a mouse model of intradermal infection, we found that Mu was initially captured by phagocytes and transported to draining lymph nodes (DLN) within host cells. Similar to Buruli ulcers in humans, the infection site eventually became ulcerated with tissue necrosis and extracellular bacteria, at later stages. In contrast to Mycobacterium bovis BCG (BCG), Mu did not disseminate to the spleen. However, mice infected with Mu or BCG developed comparable primary cellular responses to mycobacterial antigens in DLN and spleen. The role of mycolactone in this sequence of events was examined with a mycolactone-deficient (mup045) mutant of Mu. Mup045 bacilli were better internalized than wild-type (wt) bacteria by mouse phagocytes in vitro. Moreover, infection with wt but not mup045 Mu led to inhibition of TNF-alpha expression, upregulation of MIP-2 chemokine, and host cell death within 1 day. Our results suggest that mycolactone expression during the intracellular life of Mu may contribute to immune evasion by inhibiting phagocytosis, provoking apoptosis of antigen presenting cells and altering the establishment of an appropriate inflammatory reaction.

Use of a lentiviral vector encoding a HCMV-chimeric IE1-pp65 protein for epitope identification in HLA-Transgenic mice and for ex vivo stimulation and expansion of CD8(+) cytotoxic T cells from human peripheral blood cells.

H2-deleted, HLA-A2, or HLA-B7 transgenic mice were used to identify new human cytomegalovirus (HCMV)-derived major histocompatibility complex class I-restricted epitopes. Three different approaches for mice immunization were compared for their ability to induce a cytotoxic CD8(+) T cell (CTL) response: (1). inoculation of infectious HCMV, (2). injection of immunogenic synthetic peptides, and (3). infection with a newly designed HIV-derived central DNA flap positive lentiviral vector encoding the chimeric IE1-pp65 protein (Trip-IE1-pp65). Targets pulsed with either known immunogenic peptides or computer predicted ones were used to characterize CTL. Most of the mice immunized with the pp65 (495-NLVPMVATV-503) immunodominant peptide responded after one injection whereas only two of six mice responded to two successive inoculations with HCMV. Infection of mice with Trip-IE1-pp65 induced activation and expansion of CTL directed against peptides from both pp65 and IE1 and allowed identification of new epitopes. We further demonstrated the high capacity of monocyte-macrophage cells transduced with Trip-IE1-pp65 to activate and expand CTL directed against pp65 from peripheral blood mononuclear cells of HCMV-seropositive donors. Altogether these results suggest that Trip-IE1-pp65 is a powerful construct both to characterize new epitopes in combination with human leukocyte antigen-transgenic mice immunization and to provide an alternative to the use of known infectious and noninfectious approaches to expand effector T cells for adoptive immunotherapy.

Persistent, noncytolytic infection of neurons by Borna disease virus interferes with ERK 1/2 signaling and abrogates BDNF-induced synaptogenesis.

Infection of the central nervous system by Borna disease virus (BDV) provides a unique model to study the mechanisms whereby a persistent viral infection can impair neuronal function and cause behavioral diseases reminiscent of mood disorders, schizophrenia, or autism in humans. In the present work, we studied the effect of BDV infection on the response of hippocampal neurons, the main target for this virus, to the neurotrophin BDNF. We showed that persistent infection did not affect neuronal survival or morphology. However, it blocked BDNF-induced ERK 1/2 phosphorylation, despite normal expression of the TrkB BDNF receptor. In addition, BDNF-induced expression of synaptic vesicle proteins was abrogated, which resulted in severely impaired synaptogenesis and defects in synaptic organization. Thus, we provide the first evidence that a virus can interfere specifically with neurotrophin-regulated neuroplasticity, thereby hampering proper neuronal connectivity. These results may help to understand the behavioral disorders associated with BDV infection.

Dendritic cells as host cells for the promastigote and amastigote stages of Leishmania amazonensis: the role of opsonins in parasite uptake and dendritic cell maturation.

In their mammalian hosts, Leishmania are obligate intracellular parasites that mainly reside in macrophages. They are also phagocytosed by dendritic cells (DCs), which play decisive roles in the induction and shaping of T cell-dependent immune responses. Little is known about the role of DCs in the Leishmania life cycle. Here, we examined the ability of mouse bone marrow-derived DCs to serve as hosts for L. amazonensis. Both infective stages of Leishmania (metacyclic promastigotes and amastigotes) could be phagocytosed by DCs, regardless of whether they had previously been experimentally opsonized with either the complement C3 component or specific antibodies. Parasites could survive and even multiply in these cells for at least 72 hours, within parasitophorous vacuoles displaying phagolysosomal characteristics and MHC class II and H-2M molecules. We then studied the degree of maturation reached by infected DCs according to the parasite stage internalised and the type of opsonin used. The cell surface expression of CD24, CD40, CD54, CD80, CD86, OX40L and MHC class II molecules was barely altered following infection with unopsonized promastigotes or amastigotes from nude mice or with C3-coated promastigotes. Even 69 hours post-phagocytosis, a large proportion of infected DCs remained phenotypically immature. In contrast, internalisation of antibody-opsonized promastigotes or amastigotes induced DCs to mature rapidly, as shown by the over-expression of costimulatory, adhesion and MHC class II molecules. Thus, in the absence of specific antibodies (e.g. shortly after infecting naive mammals), infected DCs may remain immature or semi-mature, meaning that they are unable to elicit an efficient anti-Leishmania T cell response. Absence of DC maturation or delayed/incomplete DC maturation could thus be beneficial for the parasites, allowing their establishment and amplification before the onset of immune responses.

Constrained intracellular survival of Mycobacterium tuberculosis in human dendritic cells.

Dendritic cells (DCs) are likely to play a key role in immunity against Mycobacterium tuberculosis, but the fate of the bacterium in these cells is still unknown. Here we report that, unlike macrophages (Mphis), human monocyte-derived DCs are not permissive for the growth of virulent M. tuberculosis H37Rv. Mycobacterial vacuoles are neither acidic nor fused with host cell lysosomes in DCs, in a mode similar to that seen in mycobacterial infection of Mphis. However, uptake of the fluid phase marker dextran, and of transferrin, as well as accumulation of the recycling endosome-specific small GTPase Rab11 onto the mycobacterial phagosome, are almost abolished in infected DCs, but not in Mphis. Moreover, communication between mycobacterial phagosomes and the host-cell biosynthetic pathway is impaired, given that <10% of M. tuberculosis vacuoles in DCs stained for the endoplasmic reticulum-specific proteins Grp78/BiP and calnexin. This correlates with the absence of the fusion factor N-ethylmaleimide-sensitive factor onto the vacuolar membrane in this cell type. Trafficking between the vacuoles and the host cell recycling and biosynthetic pathways is strikingly reduced in DCs, which is likely to impair access of intracellular mycobacteria to essential nutrients and may thus explain the absence of mycobacterial growth in this cell type. This unique location of M. tuberculosis in DCs is compatible with their T lymphocyte-stimulating functions, because M. tuberculosis-infected DCs have the ability to specifically induce cytokine production by autologous T lymphocytes from presensitized individuals. DCs have evolved unique subcellular trafficking mechanisms to achieve their Ag-presenting functions when infected by intracellular mycobacteria.

In vivo protein markers of human peritoneal mesothelial cells: do they differ according to their anatomical sites?

OBJECTIVE: To highlight the in vivo protein markers in human peritoneal mesothelial cells according to their anatomical distribution and their interest in theories of peritoneal repair. DESIGN: Clinical laboratory study. SETTING: University Hospital, France. PATIENTS: Sampling of mesothelial cells during 8 laparotomies by impression smears. MAIN OUTCOME MEASURES: Immunochemical semiquantitative measures of proteins. RESULTS: There were uniform ratios of protein markers among the whole peritoneal mesothelial cell population, whatever their anatomical origin (liver, stomach, omentum, small bowel, and abdominal wall). CONCLUSION: This is consistent with the surrounding uninjured mesothelial cells or the exfoliated mesothelial cells, or both, having a role in postoperative peritoneal mesothelial repair.

Glucocorticoid treatment induces expression of small heat shock proteins in human satellite cell populations: consequences for a desmin-related myopathy involving the R120G alpha B-crystallin mutation.

A missense mutation (R120G) of the molecular chaperone alpha B-crystallin has recently been linked to a familial form of desmin-related myopathy characterized by intrasarcoplasmic aggregates of desmin. It was previously demonstrated that the mutant R120G had a defective chaperone-like function. However, the cellular and physiopathological consequences of R120G mutant expression in human muscle cells are as yet unclear. Thus, we developed a cellular model for the study of this R120G alpha B-crystallin-related desmin-related myopathy. We demonstrate that dexamethasone enhances alpha B-crystallin and HSP27 expression in normal and desmin-related myopathy-derived muscle cells. In the undifferentiated desmin-related myopathy satellite cell population no intracytoplasmic aggregates were observed. However, in differentiated satellite cells derived from a related myopathy patient, we observed an enhanced plasma membrane localization of alpha B-crystallin following glucocorticoid. We also observed that the protective effect against stress of alpha B-crystallin is altered upon glucocorticoid-induced small heat shock protein expression for the desmin-related myopathy-derived cells.