Search

Exposure of the vertebrate embryo to maternal hormones can have long-lasting effects on its phenotype, which has been studied extensively by experimentally manipulating maternal steroids, mostly androgens, in bird eggs. Yet, there is a severe lack of understanding of how and when these effects are actually mediated, hampering both underlying proximate and ultimate explanations. Here we report a novel finding that the embryo expresses androgen receptor (AR) and estrogen receptor (ERα) mRNA in its extraembryonic membranes (EMs) as early as before its own hormone production starts, suggesting a novel substrate for action of maternal hormones on the offspring. We also report the first experimental evidence for steroid receptor regulation in the avian embryo in response to yolk steroid levels: the level of AR is dependent on yolk androgen levels only in the EMs but not in body tissues, suggesting embryonic adaptation to maternal hormones. The results also solve the problem of uptake of lipophilic steroids from the yolk, why they affect multiple traits, and how they could mediate maternal effects without affecting embryonic sexual differentiation.

Ducks and chickens are hosts of avian influenza virus, each with distinctive responses to infection. To understand these differences, we characterized the innate immune response to low pathogenicity avian influenza virus H7N1 infection in chickens and ducks. Viral RNA was detected in the lungs of chickens from day 0.8 to 7, in ducks mainly at day 4. In both species viral RNA was detected in the bursa and gut. Infection in chickens resulted in up-regulation of interferon (IFN)-a and IFN-ß mRNA, while in the ducks IFN-¿ mRNA was strongly up-regulated in lung and bursa. In chickens and ducks all investigated pathogen recognition receptors (PRR) mRNA were up regulated, however, in the chicken lung Toll-like receptor (TLR)7 and melanoma differentiation-associated protein (MDA)-5 mRNA were strongly induced. TLR3, TLR7 and MDA-5 responses correlated with IFN-a and IFN-ß responses in chickens, but in ducks a correlation between IFN-a and TLR7, retinoic acid-inducible gene-I and MDA-5 was absent. We studied the responses of duck and chicken splenocytes to poly (I:C) and R848 analogues to analyze the regulation of PRRs without the interfering mechanisms of the influenza virus. This revealed IFN-a and IFN- ¿ responses in both species. MDA-5 was only strongly up-regulated in chicken splenocytes. In chickens splenocytes, time related PRR responses correlated with the IFN-a and IFN-ß response. In duck splenocytes this correlation was absent. In conclusion, chicken and duck differ in induction of MDA-5, TLR7 and IFN-a mRNA after an influenza virus infection in vivo and after in vitro stimulation with TLR antagonists.

In this study, we investigated the cell entry characteristics of dengue virus (DENV) type 2 strain S1 on mosquito, BHK-15, and BS-C-1 cells. The concentration of virus particles measured by biochemical assays was found to be substantially higher than the number of infectious particles determined by infectivity assays, leading to an infectious unit-to-particle ratio of approximately 1:2,600 to 1:72,000, depending on the specific assays used. In order to explain this high ratio, we investigated the receptor binding and membrane fusion characteristics of single DENV particles in living cells using real-time fluorescence microscopy. For this purpose, DENV was labeled with the lipophilic fluorescent probe DiD (1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt). The surface density of the DiD dye in the viral membrane was sufficiently high to largely quench the fluorescence intensity but still allowed clear detection of single virus particles. Fusion of the viral membrane with the cell membrane was evident as fluorescence dequenching. It was observed that DENV binds very inefficiently to the cells used, explaining at least in part the high infectious unit-to-particle ratio. The particles that did bind to the cells showed different types of transport behavior leading to membrane fusion in both the periphery and perinuclear regions of the cell. Membrane fusion was observed in 1 out of 6 bound virus particles, indicating that a substantial fraction of the virus has the capacity to fuse. DiD dequenching was completely inhibited by ammonium chloride, demonstrating that fusion occurs exclusively from within acidic endosomes. Dengue virus (DENV) is an enveloped, positive-strand RNA virus belonging to the family Flaviviridae, which also includes tick-borne encephalitis virus, yellow fever virus, and West Nile virus. Flavivirus virions contain three structural proteins: the C (capsid) protein, the M (membrane) protein, and the E (envelope) protein (12, 26). Multiple copies of the C protein associate with the viral RNA to form the nucleocapsid (26). The nucleocapsid is surrounded by a lipid bilayer in which the M and E glycoproteins are inserted. In the infected cell, the M protein is produced as a precursor protein called prM, which is believed to function as a chaperone during the folding

Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of "toxin-coated bacteria" proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or "free" in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. Technical and human support provided by SGIker (Analytical and High-Resolution Microscopy in Biomedicine Service of UPV/EHU) and Rocio Alonso for excellent technical assistance are gratefully acknowledged. We also thank Dr. N. Guiso from the Pasteur Institute (Paris, France) for the Bordetella pertussis strains BP18323 and non-virulent bgv negative BP18323H-. This study was supported by grants from the Spanish Ministerio de Ciencia y Tecnologia (Project BFU 2012-36241), and the Basque Government (Grupos Consolidados IT849-13 and ETORTEK Program). K.B.U and D.G.B. were recipients of a fellowship from the Bizkaia Biophysics Foundation. A.E.G. was a recipient of a research contract (INNPACTO 2010) and A.E.L. was a recipient of a fellowship from the University of the Basque Country (UPV/EH). A patent with reference N/Ref.: P1683USPC (2004) was approved.

The type VI secretion system (T6SS) of Gram-negative bacteria has been involved in various processes, notably bacterial competition and eukaryotic cell subversion. Most Pseudomonas aeruginosa strains possess three T6SS gene clusters, but only the function of the first T6SS (H1-T6SS) has been clearly elucidated. It is involved in the secretion of three toxins (Tse1 to -3) that target bacterial competitors. In the case of the H2- and H3-T6SS, no clear function has been assigned, and only one effector has been associated with these systems. Yet the H2-T6SS was proposed to promote P. aeruginosa internalization in nonphagocytic epithelial cells. Although the H2-T6SS genetic organization is conserved across P. aeruginosa isolates, one feature is the presence of an additional transcriptional unit in the PA14 strain H2-T6SS cluster, which is divergent from the core H2-T6SS genes. A specific set of four genes encodes an Hcp protein (Hcp2), a VgrG protein (VgrG14), an Rhs element (PA14_43100 or RhsP2), and a protein with no homologies with previously characterized proteins (PA14_43090). In this study, we engineered a P. aeruginosa PA14 strain carrying an arabinose-inducible H2-T6SS on the chromosome. We showed that arabinose induction readily promotes assembly of the H2-T6SS, as seen by monitoring Hcp2 secretion. We further studied the secretion fate of VgrG14 and RhsP2, but these were not detectable in the extracellular medium. We finally investigated whether activation of the PA14 H2-T6SS gene cluster could influence phenotypic traits such as internalization in eukaryotic cells, and we reported noteworthy differences compared to strain PAO1, which may be accounted for by the described genetic differences.

Natural killer (NK) cell activity is conserved throughout vertebrate development, but characterization of non-mammalian NK-cells has been hampered by the absence of specific mAbs for these cells.Monoclonal antibodies were generated against in vitro IL-2 expanded sorted CD3-CD8 alpha+ peripheral blood lymphocytes, previously described to contain chicken NK-cells. Screening of embryonic and adult splenocytes with hybridoma supernatants resulted in five candidate NK markers.Activation of chicken NK-cells with PMA/Ionomycin or with the NK target cell-line LSCC-RP9 resulted in increased expression of CD107 (LAMP-1) and a newly developed flow cytometry based cytotoxicity assay showed that NK-cells were able to kill target cells. Combining NK markers with functional assays indicated that marker positive cells showed NK-cell function.In conclusion, we generated new monoclonal antibodies and developed two functional assays which will enhance our understanding of the role of NK-cells in healthy and diseased chickens. (C) 2010 Elsevier Ltd. All rights reserved.

Studies utilizing various immunodeficient mouse models of rotavirus (RV) infection demonstrated significant roles of RV-specific secretory immunoglobulin A (IgA), CD4+T cells, and CD8+T cells in the clearance of RV and protection from secondary infection. Secretion of small but detectable amounts of IgA in RV-infected αβ T-cell receptor knockout mice (11) and distinctive anatomical localization and physiology of B1 cells suggested that B1 cells might be capable of producing RV-specific intestinal IgA in a T-cell-independent fashion and, therefore, be responsible for ablation of RV shedding. We investigated the role of B1 cells in the resolution of primary RV infection using a SCID mouse model. We found that the adoptive transfer of unseparated peritoneal exudate cells ablates RV shedding and leads to the production of high levels of RV-specific intestinal IgA. In contrast, purified B1 cells do not ablate RV shedding and do not induce a T-cell-independent or T-cell-dependent, RV-specific IgA response but do secrete large amounts of polyclonal (total) intestinal IgA. Cotransfer of mixtures of purified B1 cells and B1-cell-depleted peritoneal exudate cells differing in IgA allotypic markers also demonstrated that B2 cells (B1-cell-depleted peritoneal exudate cells) and not B1 cells produced RV-specific IgA. To our knowledge, this is the first observation that B1 cells are unable to cooperate with CD4+T cells and produce virus-specific intestinal IgA antibody. We also observed that transferred CD4+T cells alone are capable of resolving RV shedding, although no IgA is secreted. These data suggest that RV-specific IgA may not be obligatory for RV clearance but may protect from reinfection and that effector CD4+T cells alone can mediate the resolution of primary RV infection. Reconstitution of RV-infected SCID mice with B1 cells results in the outgrowth of contaminating, donor CD4+T cells that are unable to clear RV, possibly because their oligoclonal specificities may be ineffective against RV antigens.