Presence of dendritic cells in chicken spleen cell preparations and their functional interaction with the parasite Toxoplasma gondii.

Toxoplasmosis is a worldwide epizootic disease of mammals. Chickens, albeit being less susceptible, can be contaminated in free-range flocks and may have an important role in parasite transmission. Plastic adherence selection of chicken spleen cells enriched 8F2+ (putative chicken CD11c) MHC II+ cells of the myeloid type; however, we did not succeed to separate dendritic cells from macrophages using their feature to become loosely adherent after culture as in mammals. Still we clearly identified dendritic-like cells being morphologically distinguishable from macrophages in the KUL01 (macrophage marker) negative fraction, exhibiting responsiveness to LPS and parasite extracts by developing characteristic cellular protrusions as well as a minor phagocytic incorporation of dead parasites. Live T. gondii tachyzoites were able to invade the two different types of myeloid adherent cells, to replicate, and to induce an overall decrease in the expression of MHC II and co-stimulatory molecules, CD80 and CD40. Our data indicate that dendritic cells in addition to macrophages may have a role in hiding viable replicating T. gondii tachyzoites from the immune system and in shuttling them to different organs in the chicken as previously described for different Apicomplexa infecting mammals.

Metastatic pancreatic cancer is dependent on oncogenic Kras in mice.

Pancreatic cancer is one of the deadliest human malignancies, and its prognosis has not improved over the past 40 years. Mouse models that spontaneously develop pancreatic adenocarcinoma and mimic the progression of the human disease are emerging as a new tool to investigate the basic biology of this disease and identify potential therapeutic targets. Here, we describe a new model of metastatic pancreatic adenocarcinoma based on pancreas-specific, inducible and reversible expression of an oncogenic form of Kras, together with pancreas-specific expression of a mutant form of the tumor suppressor p53. Using high-resolution magnetic resonance imaging to follow individual animals in longitudinal studies, we show that both primary and metastatic lesions depend on continuous Kras activity for their maintenance. However, re-activation of Kras* following prolonged inactivation leads to rapid tumor relapse, raising the concern that Kras*-resistance might eventually be acquired. Thus, our data identifies Kras* as a key oncogene in pancreatic cancer maintenance, but raises the possibility of acquired resistance should Kras inhibitors become available for use in pancreatic cancer.

Immunological responses induced by a DNA vaccine expressing RON4 and by immunogenic recombinant protein RON4 failed to protect mice against chronic toxoplasmosis.

The development of an effective vaccine against Toxoplasma gondii infection is an important issue due to the seriousness of the related public health problems, and the economic importance of this parasitic disease worldwide. Rhoptry neck proteins (RONs) are components of the moving junction macromolecular complex formed during invasion. The aim of this study was to evaluate the vaccine potential of RON4 using two vaccination strategies: DNA vaccination by the intramuscular route, and recombinant protein vaccination by the nasal route. We produced recombinant RON4 protein (RON4S2) using the Schneider insect cells expression system, and validated its antigenicity and immunogenicity. We also constructed optimized plasmids encoding full length RON4 (pRON4), or only the N-terminal (pNRON4), or the C-terminal part (pCRON4) of RON4. CBA/J mice immunized with pRON4, pNRON4 or pCRON4 plus a plasmid encoding the granulocyte-macrophage-colony-stimulating factor showed high IgG titers against rRON4S2. Mice immunized by the nasal route with rRON4S2 plus cholera toxin exhibited low levels of anti-RON4S2 IgG antibodies, and no intestinal IgA antibodies specific to RON4 were detected. Both DNA and protein vaccination generated a mixed Th1/Th2 response polarized towards the IgG1 antibody isotype. Both DNA and protein vaccination primed CD4+ T cells in vivo. In addition to the production of IFN-γ, and IL-2, Il-10 and IL-5 were also produced by the spleen cells of the immunized mice stimulated with RON4S2, suggesting that a mixed Th1/Th2 type immune response occurred in all the immunized groups. No cytokine was detectable in stimulated mesenteric lymph nodes from mice immunized by the nasal route. Immune responses were induced by both DNA and protein vaccination, but failed to protect the mice against a subsequent oral challenge with T. gondii cysts. In conclusion, strategies designed to enhance the immunogenicity and to redirect the cellular response towards a Th1 type response against RON4 could lead to more encouraging results.

Dendritic cells loaded with HIV-1 p24 proteins adsorbed on surfactant-free anionic PLA nanoparticles induce enhanced cellular immune responses against HIV-1 after vaccination.

Biodegradable nanoparticles with surface adsorbed antigens represent a promising method for in vivo delivery of vaccines targeting a wide range of infectious diseases or cancers. We investigated the feasibility of loading dendritic cells with a vaccine antigen, HIV p24 protein, on the surface of surfactant-free anionic (d,l-lactic acid, PLA) nanoparticles. The p24 protein had a high affinity for the nanoparticles and the antigenicity and immunogenicity of the p24 protein on the nanoparticle was well preserved after immunization. p24-coated nanoparticles were efficiently taken up by mouse dendritic cells (DCs), inducing DC maturation by increasing MHC-I, MHC-II, CD40, CD80 and CD86 surface expression and secreting IL-12 (p70) and IL-4. We evaluated the ability of DCs pulsed with p24-coated nanoparticles to elicit an optimal humoral and cellular immune response in the blood and intestine. DCs pulsed with p24-nanoparticles induced high seric and mucosal antibody production and elicited strong systemic and local lymproliferative responses, correlated with a Th1/Th2-type response, and systemic CTL responses in mice. Thus, DCs pulsed with antigen-loaded PLA nanoparticles may provide a novel delivery tool for cell therapy vaccination against chronic infectious diseases.

Exosomes are an effective vaccine against congenital toxoplasmosis in mice.

Toxoplasmosis is a serious disease in humans and may cause abortion or congenital infection if a woman is exposed to the disease for the first time during pregnancy. Infection before pregnancy normally results in immunity protecting the foetus, suggesting that it may be possible to block vertical transmission of the parasite by appropriate vaccination before pregnancy. We found that the vaccination of CBA/J mice, before pregnancy, with exosomes secreted by SRDCs pulsed in vitro with Toxoplasma gondii-derived antigens (TAg) induced a protective response in the pups. Indeed, vaccination resulted in the presence of significantly fewer cysts in pup brains. This protection was associated with strong humoral responses in the serum in vivo. We also observed cellular responses in vivo, with cell proliferation associated with the production of cytokines by the splenocytes. Thus, exosomes are nucleic acid-free vesicles able to induce immune responses correlated with protection against T. gondii infection in a congenital model. They are therefore a potentially useful tool for vaccination against infectious disease.

Generation of specific Th1 and CD8+ T-cell responses by immunization with mouse CD8+ dendritic cells loaded with HIV-1 viral lysate or envelope glycoproteins.

Immunization with antigen-pulsed dendritic cells (DCs) can be used to elicit optimal immune responses. We developed the SRDC cell line, with a morphology, phenotype and activity similar to mouse splenic CD4(-)CD8alpha(+)CD205(+)CD11b(-) dendritic cells, which induce a polarized Th1 immune response. We evaluated the ability of SRDCs pulsed with HIV-1 viral lysate, oligomeric soluble gp140 or capsid p24 to induce specific antibody and T-cell responses in CBA/J mice. Immunization with all loaded SRDCs elicited antibody responses against the antigens tested. However, only HIV-1 viral lysate and gp140-pulsed SRDCs elicited specific CD4(+) and CD8(+) T-cell responses. These findings demonstrate the value of well characterized DC lines for optimizing the antigen-loading mixture, according to the DC population targeted. Our data suggest that splenic DCs pulsed with complex antigens, such as HIV-1 viral lysate or oligomeric soluble gp140, could be used as vaccines, eliciting strong primary Th1-polarized and humoral immune responses against HIV proteins in vivo.