The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry.

The complexity of signal transduction resulting from the contact of human immunodeficiency virus type 1 (HIV-1)-infected cells and mucosal cells has hampered our comprehension of HIV-1 mucosal entry. Such process is driven efficiently only by viral synapse contacts, whereas cell-free HIV-1 remains poorly infectious. Using CD4+ T-cells expressing only HIV-1 envelope inoculated on human adult foreskin tissues, we designed methodologies to identify the signals transduced in foreskin keratinocytes following HIV-1-envelope-dependent viral synapse formation. We find that the viral synapse activates the MyD88-independent TLR-4-nuclear factor (NfκB) signaling pathway in keratinocytes and the subsequent secretion of cytokines including thymic stromal lymphopoietin (TSLP), a cytokine linking innate and T-helper type 2-adaptive immune responses. Moreover, the viral synapse upregulates the non-coding microRNA miR-375, known to control TSLP, and transfection of keratinocytes with anti-miR-375 blocks significantly TSLP secretion. Thus, the secretion of TSLP by keratinocytes is induced by the viral synapse in a miR-375 controlled manner. At the tissue level, these signals translate into the epidermal redistribution of Langerhans cells and formation of conjugates with T-cells, recapitulating the initial events observed in human foreskin infection by HIV-1. These results open new possibilities for designing strategies to block mucosal HIV-1 transmission, the major pathway by which HIV-1 spreads worldwide.

Calcitonin gene-related peptide inhibits human immunodeficiency type 1 transmission by Langerhans cells via an autocrine/paracrine feedback mechanism.

AIM: Peripheral neurones innervating mucosal epithelia are in direct contact with resident immune cells, including Langerhans cells (LCs). Such neurones secrete the neuropeptide calcitonin gene-related peptide (CGRP) that modulates LCs function. We recently found that CGRP strongly inhibits human immunodeficiency virus type 1 (HIV-1) transmission, by interfering with multiple steps of mucosal LC-mediated HIV-1 transfer, including increased expression of the LC-specific lectin langerin. Herein, we investigated the anti-HIV-1 mechanism of CGRP. METHODS: In the presence of CGRP, HIV-1 transfer from LCs to CD4+ T cells was tested with viral clones using either the HIV-1 co-receptor CCR5 (R5) or CXCR4 (X4). Surface expression of CCR5, CXCR4 and langerin was evaluated by flow cytometry. CGRP secretion by LCs was measured with an enzyme immunoassay. Expression of the multimeric CGRP receptor was examined by quantitative real-time RT-PCR and immuno-fluorescent microscopy. RESULTS: Calcitonin gene-related peptide decreased transfer of HIV-1 R5, but increased that of X4. These opposing effects correlated with decreased CCR5 vs. increased CXCR4 surface expression in LCs. Inhibition of HIV-1 R5 transfer by CGRP involved signal transducer and activator of transcription 4 (STAT4) activation. Both αCGRP and ßCGRP were similarly efficient in decreasing HIV-1 R5 transfer and increasing langerin expression. LCs secreted low basal levels of endogenous CGRP, which increased markedly following CGRP treatment. CGRP also increased expression of its cognate receptor in LCs. CONCLUSION: CGRP engages a positive feedback mechanism that would further enhance its anti-HIV-1 activity. This information might be relevant for the therapeutic use of CGRP as a prophylactic agent against HIV-1.

The adult penile urethra is a novel entry site for HIV-1 that preferentially targets resident urethral macrophages.

The penile urethra is routinely targeted by sexually transmitted bacterial and viral pathogens, and also represents a probable site for HIV type-1 (HIV-1) entry. Yet, the mechanisms of urethral HIV-1 transmission are unknown. To describe the initial steps of penile HIV-1 entry, we obtained whole penile tissues from individuals undergoing elective gender reassignment and developed ex vivo polarized explants of different penile epithelia, as well as in vitro immunocompetent reconstructed urethra. In penile explants, 1 h exposure to cell-associated HIV-1 results in higher HIV-1 entry into the urethra, whereas the fossa navicularis and glans are relatively resistant to HIV-1. CCR5+/CD4+ urethral macrophages are the initial cells infected by HIV-1, which exit the epithelial compartment following inoculation with cell-associated HIV-1 that induces decreased CCL2/MCP-1 production. Urethral T cells are mostly CD8+ or naive CD4+, and not infected by HIV-1 on its early entry. In urethral reconstructions, efficient translocation of cell-associated HIV-1 depends on viral tropism (R5>X4) and can be decreased by gp41-specific IgAs. Cell-free HIV-1 is inefficient at urethral penetration. Our results identify the male urethra as a novel entry site for HIV-1 that targets resident urethral macrophages. These results might explain the incomplete prophylactic efficacy of male circumcision in reducing HIV-1 transmission.

Virus like particle based strategy to elicit HIV-protective antibodies to the alpha-helic regions of gp41.

Natural antibodies to gp41 inhibit HIV-1 replication through the recognition of two different regions, corresponding to the leucine zipper motif in the HR1 alpha-helix and to another motif within HR2 region, hosting 2F5 and 4E10 epitope. This study aimed at reproducing such protective responses through VLP vaccination. Six regions covering the alpha-helical regions of gp41 were conjugated to the surface of AP205 phage-based VLPs. Once administered in mice via systemic or mucosal route, these immunogens elicited high titers of gp41-specific IgG. Immunogenicity and HIV infectivity reduction were obtained either with HR2 regions or with peptides where aminoacid strings were added to either the C-terminus or N-terminus of core epitope in HR1 region. Antibody-dependent cell cytotoxicity (ADCC) activity was induced by one of the HR2 epitopes only. These results may have relevant implications for the development of new vaccinal approaches against HIV infection.

Generation of HIV-1 Virus-Like Particles expressing different HIV-1 glycoproteins.

Elicitation of a potent and broadly neutralizing antibody response is the main goal of an effective preventive HIV-1 vaccine. It has been shown by us and others that the expression of Env glycoproteins on the surface of particulate structures, such as Virus-Like Particles (VLPs), could be a more efficient strategy to deliver conformational epitopes to the immune system. To this aim, VLPs expressing native HIV Env gp140 or gp41 glycoproteins have been produced in insect cells using a baculovirus expression system and characterized for appropriate protein expression. VLP-bound HIV gp140 glycoprotein showed the appropriate expression and trimeric conformation. Immunogenicity studies have been performed in BALB/C mice by intra-peritoneal administration and sera from immunized mice have been tested in ELISA assays, for their reactivity with HIV specific antigens, as well as in ex vivo neutralization assay. Sera from immunized animals showed a high reactivity with individual HIV proteins expressed in VLPs. Results of TZM-bl based neutralization assay show that combined sera from animals independently immunized with gp140- or full-length-gp41-expressing VLPs have an additive/synergistic effect in the neutralization activity of HIV pseudoviruses. In conclusion, novel VLPs expressing different HIV Env glycoproteins with native trimeric conformation have been generated, showing the induction of effective antibody response with neutralization activity in TZM-bl neutralization assay. These results confirm the effectiveness of VLPs as presentation and delivery system for conformational proteins and show the improved neutralization activity upon the combination of anti-sera elicited by different HIV envelope antigens, suggesting the possibility of broadening the spectrum of viral epitopes targeted by immune response.

Within 1 h, HIV-1 uses viral synapses to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T cell conjugates.

Although circumcision reduces male acquisition of human immunodeficiency virus type-1 (HIV-1) by 60%, the initial mechanisms of HIV-1 transmission at the foreskin remain elusive. We have established two novel and complementary models of the human adult foreskin epithelium, namely, ex vivo foreskin explants and in vitro reconstructed immunocompetent foreskins. In these models, efficient HIV-1 transmission occurs after 1 h of polarized exposure of the inner, but not outer, foreskin to mononuclear cells highly infected with HIV-1, but not to cell-free virus. HIV-1-infected cells form viral synapses with apical foreskin keratinocytes, leading to polarized budding of HIV-1, which is rapidly internalized by Langerhans cells (LCs) in the inner foreskin. In turn, LCs migrate toward the epidermis-dermis interface to form conjugates with T cells, thereby transferring HIV-1. Seminal plasma mixed with cervicovaginal secretions inhibits HIV-1 translocation. This set of results rationalizes at the cellular level the apparent protective outcome of circumcision against HIV-1 acquisition by men.

HIV-1 gp41-specific monoclonal mucosal IgAs derived from highly exposed but IgG-seronegative individuals block HIV-1 epithelial transcytosis and neutralize CD4(+) cell infection: an IgA gene and functional analysis.

AIDS is mainly a sexually transmitted disease, and accordingly, mucosal tissues are the primary sites of natural human immunodeficiency virus type-1 (HIV-1) transmission. Mucosal immunoglobulin A (IgA) antibody specific for HIV-1 envelope gp41 subunit is one correlate of protection in individuals who are highly sexually exposed to HIV-1 but remain persistently IgG seronegative (HEPS). Understanding these peculiar IgAs at the gene and functional level is possible only with monoclonal IgAs. We have constructed a mucosal Fab IgA library from HEPS and have characterized a series of HIV-1 IgAs specific for gp41 that, in vitro, are transcytosis-blocking and infection-neutralizing. Characterization of their IgA genes shows that Fab specific for the gp41 membrane-proximal region harbors a long heavy-chain CDR3 loop (CDRH3) similar to the two broadly neutralizing IgG monoclonal antibodies, 2F5 and 4E10. Furthermore, the selected Fab IgA shows extensive somatic mutations that cluster in the CDR regions, indicating that affinity maturation due to an antigen-driven process had occurred in HEPS individuals, presumably upon multiple exposures to HIV. This analysis of HEPS monoclonal IgA gives a unique opportunity to correlate an antibody function (resistance to a pathogen in vivo) with an antibody gene. Such neutralizing monoclonal IgAs could be used in microbicide formulation.

Cyclic FEE peptide increases human gamete fusion and potentiates its RGD-induced inhibition.

BACKGROUND: Alpha6beta1 integrin has been proposed to act as a sperm receptor on the mouse oocyte by interacting with spermatozoon fertilin beta. We investigated, in humans, whether oocyte integrins could act similarly in gamete fusion, using a cyclic peptide containing the putative disintegrin-binding domain of human fertilin beta [cyclic FEE (cFEE)] and RGD peptide. METHODS: Zona-free eggs were inseminated in the absence or presence of peptides. To maintain the membrane protein pattern, the zona pellucida was removed by microdissection. Immunofluorescence and confocal microscopy were used to detect integrin subunits on the oocyte. RESULTS: Unexpectedly, cFEE alone increased human gamete fusion by 94% instead of inhibiting fertilization. Furthermore, cFEE together with RGD potentiated the RGD-induced inhibition of fertilization in a dose-dependent manner. The data suggested the hypothesis of integrin cross-talk, further supported by the co-localization of alpha6beta1 and alphavbeta3 integrins, the putative receptors of cFEE and RGD peptides, respectively. CONCLUSIONS: RGD-sensitive and -insensitive integrins may be associated in a multimolecular complex working as a sperm receptor on the human oocyte membrane. Supplementation of human IVF culture medium with cFEE peptide might improve fertilization rates in ART.

Annexin expressions are temporally and spatially regulated during rat hepatocyte differentiation.

Annexin (Anx) 1, 2, 5, and 6 expressions were determined at the transcriptional and translational levels in the rat hepatocytes from gestational day 15 to postnatal day 17. Dramatic shifts were observed in Anx 1 and 2 levels, which peaked at day 1 and gestational day 20, respectively, and reached low levels thereafter. However, Anx 5 and 6 rates were more constant. Prenatal administration of dexamethasone (dex) resulted in a decrease of Anx 1 mRNA levels, and a strong increase in Anx 2 mRNA contents. In adult hepatocytes cultured in the presence of EGF or HGF, Anx 1 and 2 expressions resumed. By immunohistochemistry, Anx 1 was detected only in the cytoplasm of hepatocytes of 1- to 3-day-old rats, Anx 2 and 6 both exhibited a redistribution from the cytoplasm toward the plasma membrane, and Anx 5 was present in the nucleus, cytoplasm, and plasma membrane. Thus, Anx 1, 2, 5, and 6 have individual modes of expression and localization in the differentiating hepatocytes, where they might play unique roles at well defined phases of liver ontogeny.

Mouse offspring after microinjection of heated spermatozoa.

The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56 degrees C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.

Bioelectric recognition assay (BERA).

A novel biosensory method has been developed for the determination of various chemical and biological molecules by assessing their electrophysiological interactions with a group of cells and cell components immobilized in a gel matrix that preserves their 'physiological' functions. The method was applied for the detection of: (i) hepatitis C virus in human blood samples; (ii) plant viruses; and (iii) a herbicide (glyphosate) in aqueous solutions. It was able to rapidly (assay time 3-5 min) and specifically detect the molecules in question at a concentration lower than 100 pg/ml, among other compounds f similar structure. The potential use of BERA biosensors for a rapid and cost-efficient molecule determination without prior knowledge of a specific receptor-molecule interaction is discussed.

Secretory IgA specific for a conserved epitope on gp41 envelope glycoprotein inhibits epithelial transcytosis of HIV-1.

As one of the initial mucosal transmission pathways of HIV (HIV-1), epithelial cells translocate HIV-1 from apical to basolateral surface by nondegradative transcytosis. Transcytosis is initiated when HIV-1 envelope glycoproteins bind to the epithelial cell membrane. Here we show that the transmembrane gp41 subunit of the viral envelope binds to the epithelial glycosphingolipid galactosyl ceramide (Gal Cer), an alternative receptor for HIV-1, at a site involving the conserved ELDKWA epitope. Disrupting the raft organization of the Gal Cer-containing microdomains at the apical surface inhibited HIV-1 transcytosis. Immunological studies confirmed the critical role of the conserved ELDKWA hexapeptide in HIV-1 transcytosis. Mucosal IgA, but not IgG, from seropositive subjects targeted the conserved peptide, neutralized gp41 binding to Gal Cer, and blocked HIV-1 transcytosis. These results underscore the important role of secretory IgA in designing strategies for mucosal protection against HIV-1 infection.

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection.

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.

B7 cosignal potentiates apoptosis of uninfected CD4+ T lymphocytic cell lines primed by HIV envelope proteins.

In lymphoid organs, follicular dendritic cells (FDCs), monocytes, and macrophages are targets for HIV infection and reservoirs for infectious virus. Strikingly, the apoptotic cells in these sites are essentially uninfected CD4+ T lymphocytes, but lie in close proximity to infected cells or FDCs carrying trapped HIV virions. To decipher this apoptotic pathway, we have established a two-step experimental system that reproduces in vitro the HIV envelope protein-mediated apoptosis restricted to uninfected CD4+ T lymphocytic cell lines. In this assay, uninfected CD4+ T cell targets undergo apoptosis following an initial priming step on HeLa cells expressing functional HIV envelope proteins at their plasma membrane and a second and necessary stimulation step via the CD3-TCR complex. The CD4+ T lymphocytic cells susceptible to apoptosis are, in contrast, resistant to cell fusion mediated by HIV envelope protein and express SDF-1. FDCs and macrophages are known to be high B7 expressors. Thus in lymph nodes, the cells that have trapped HIV particles in immune complexes at the plasma membrane present both HIV envelope proteins and B7.1 at their surface. We mimicked this situation in vitro by priming CD4+ T lymphocytes on cells expressing the costimulatory molecule B7 in addition to HIV envelope proteins, and show that it resulted in an acceleration and a twofold increase in apoptosis. Finally, we characterized two enzymes, PI3Kinase and PI-PLC, which are both downstream effectors of the CD4 (HIV envelope protein receptor) and CD28 (B7 receptor) activation pathways, and that participated in the early steps of priming for apoptosis.

Infectious human immunodeficiency virus can rapidly penetrate a tight human epithelial barrier by transcytosis in a process impaired by mucosal immunoglobulins.

Mucosal surfaces are the main natural site of entry into the body for human immunodeficiency virus (HIV). Herein, an alternative mechanism for virus spread is described. The mechanism, which involves transcytosis of endosome-internalized HIV-particles, was generated by contact of HIV-infected cells with the apical surface of an epithelial cell line. Transcytosed viruses rapidly (in 20-30 min) access the serosal side of the epithelial barrier without infecting the epithelium itself. In turn, transcytosed HIV could infect host submucosal mononucleated target cells, and thus the infection could spread. An investigation was done to determine whether mucosal antibodies could block HIV transcytosis. Both secretory IgA (S-IgA) and IgG that were purified from colostrum from HIV-seropositive women impaired HIV transcytosis, irrespective of the level of the recombinant HIV envelope anti-gp160-specific activities in an ELISA. However, specific S-IgAs were more efficient than IgG. Therefore, mucosal-specific S-IgA to HIV-1 could be relevant to reducing infectivity of HIV-1 in corporeal fluids.

Intracellular neutralization of HIV transcytosis across tight epithelial barriers by anti-HIV envelope protein dIgA or IgM.

Human immunodeficiency virus, generated during contact between HIV-infected cells and the apical surface of an epithelial cell, can cross a tight epithelial barrier by transcytosis. We show that transcytosis of primary HIV isolates is blocked by dimeric IgA or IgM against HIV envelope proteins. Neutralization occurs intracellularly within the apical recycling endosome, and immune complexes are specifically recycled to the mucosal surface. One epitope involved in neutralization is a conserved sequence of the gp41 HIV envelope protein subunit. Finally, transcytosis also occurs across functional human mucosal tissue in a process inhibited by a serosal internalization of IgM against the HIV envelope protein. These results suggest that induction of mucosal immunity to HIV envelope proteins may impair the transcytotic route of HIV mucosal transmission.

Dictyostelium discoideum cells shed vesicles with associated DNA and vital stain Hoechst 33342.

Dictyostelium discoideum cells are highly resistant to xenobiotics. We previously observed that these primitive eukaryotic cells contain a 170-kDa P-glycoprotein, mediating multidrug resistance in mammalian cells, but nonfunctional in Dictyostelium cells. We show here that D. discoideum cells vitally stained with the DNA-specific dye, Hoechst 33342, release fluorescent material in their culture medium. Electron microscopy and lipid analysis demonstrate the vesicular nature of this material. Moreover, nucleic acids associate with these extracellular vesicles independently of Hoechst vital staining. The main vesicular DNA component exhibits a size > 21 kb. Shedding of microvesicles during cell growth is not concomitant with programmed cell death. We propose that these extracellular vesicles are involved in a new cellular resistance mechanism against xenobiotics. Furthermore, since the association of DNA with vesicles occurs in physiological growth conditions and independently of vital staining, the new shedding process might be involved in a more general intercellular mechanism.

Human gamete fusion can bypass beta1 integrin requirement.

Since alpha6beta1 integrin has been shown to function as a sperm adhesion receptor in the mouse, we investigated the potential role of beta1 integrin in the gamete fusion process in humans. The expression of beta1 integrin was morphologically analysed by indirect immunofluorescence and confocal microscopy. A homogeneous and intense staining was detected at the plasma membrane, and in some subcortical vesicles of germinal vesicle stage oocytes (GV). Beta1 almost disappeared from oolemma and cytoplasm of metaphase I (MI) oocytes, but was re-expressed as asymmetrical patches at the plasma membrane of metaphase II stage oocytes (MII). A functional fusion assay based on Hoechst or calcein-AM dye transfer from one gamete to the other showed that maturing oocytes were able to fuse with an increasing number of spermatozoa (11-22 from GV to MII respectively), and that fused spermatozoa co-localized with beta1 integrin patches. Human gamete fusion was only partially inhibited either by RGD-containing peptide (GRGDTP), or by blocking anti-human beta1 integrin monoclonal antibody (DE9), with a maximum of 50% inhibition. Despite the combined addition of GRGDTP and blocking mouse anti-human beta1 integrin DE9 in the assay, a complete inhibition of fusion could not be achieved. A mouse polyclonal antibody raised against human oocyte membranes was more potent in inhibiting the fusion. Since beta1 integrin expression at the plasma membrane was not correlated to oocyte fusibility, and since it was only partially inhibited by DE9 and/or RGD peptide, we suggest that human gamete fusion can bypass the beta1 requirement. Beta1 integrin certainly participates in human gamete fusion by acting in co-operation with multiple integrin/disintegrin couples or another cofactor, not yet identified.

In adrenocortical tissue, annexins II and VI are attached to clathrin coated vesicles in a calcium-independent manner.

We have previously characterized three populations of clathrin coated vesicles (CCVs) isolated from bovine adrenocortical tissue and designated them as large, medium and small coated vesicles, i.e., LCV, MCV and SCV, respectively. Here, we show that annexins II and VI, two of the annexins involved in membrane traffic, are present in the three populations of CCVs but with different distributions between coat proteins (CP) and lipidic vesicle membrane. Annexin VI is only associated with the membrane, whatever the CCV population. In contrast, annexin II is differently distributed between coat and membrane, depending on the CCV population. Both annexins are bound to membranes in a calcium-independent manner and solubilization studies in Triton X114 (TX114) suggest that they interact poorly with lipids by hydrophobic interactions. Ligand blotting experiments show that both annexins bind to CCV proteins: annexin II to a 200-kDa component in all CCVs and annexin VI to a 100-kDa component in LCV and SCV identified as dynamin, a GTPase essential for endocytic CCV pinching off. Dynamin is tightly associated to annexin VI only in LCVs, the endocytic [transferrin (Tf) positive] vesicles. Our data suggest that annexins II and VI could define specific protein-lipid interaction microdomains that could play a role in the different functions of the CCVs.