OR.70. Humanized Mice to Test Influenza Vaccines
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Cryptococcus neoformans is an opportunistic yeast that causes meningoencephalitis and significant mortality in patients with impaired immune systems. Several yeast cell wall proteins are recognized by the innate immune system in mice and humans, however the molecular mechanisms and receptors used by immune cells to bind and trigger cell activation to C. neoformans have not been fully elucidated. Using a high-throughput RNAi screen we found that CD36, a class B scavenger receptor, mediates macrophage activation to C. neoformans stimulation. Here we report that macro- phages isolated from CD36-/- mice had marked reduction in binding and phagocytosis to C. neoformans and to C. neoformans-induced production of cytokines and chemo- kines in vitro. In addition, we show that CD36 collaborates with TLR2 for C. neoformans-induced cytokine and chemo- kine production. Finally, we found that the number of yeast cells in the liver and spleen of C. neoformans infected CD36-/- mice were significantly higher than wild-type mice. In contrast, we found that the expression of proinflammatory cytokines and chemokines in the organs of C. neoformans infected CD36-/- mice were significantly reduced than wild- type mice. Together these data demonstrate that CD36 plays a significant role in the recognition of C. neoformans by mediating binding and proinflammatory cytokine production. doi:10.1016/j.clim.2008.03.076 Fighting Pathogens Sunday, June 8 2:45 pm–4:45 pm OR.70. Humanized Mice to Test Influenza Vaccines Chun Yu, Mike Gallegos, Florentina Marches, Sandra Zurawski, Gerard Zurawski, Jacques Banchereau, A. Karolina Palucka. Baylor Institute for Immunology Research, Dallas, TX Critical to the development of novel vaccines is the availability of in vivo models of the human immune sys- tem that permit testing of vaccine efficacy. Here, we used NOD/SCID beta2m-/- immunodeficient mice which, when engrafted with human CD34+ hematopoietic progenitors, develop all subsets of human dendritic cells (DCs) and B cells. T cells and their subsets can be reconstituted by adoptive transfer. Humice can develop Influenza-specific immunity upon vaccination with ex vivo generated DCs. Vaccination with heat-inactivated Influenza virus pulsed DCs leads to the expansion of FluM1 tetramer binding CD8+ T cells and the development of Influenza-specific immunoglobulins in the serum. Importantly, influenza-specific immunoglobulins are protective and can inhibit Influenza virus-induced hemag- glutination. We also show that these mice can generate recall CD8+ T cell responses upon exposure to seasonal influenza virus vaccines: live attenuated trivalent vaccine, i.e. FluMist; or killed trivalent vaccine, i.e., Fluzone. CD8+ T cells specific to two influenza antigens FluM1 and NS1 can be detected in the blood of mice vaccinated with FluMist but not in mice vaccinated with Fluzone. Specific T cells are also present in the spleen and peripheral tissues (lung) demon- strating that human T cells can utilize murine signals for trafficking and extravasation. Upon short-term ex vivo antigen exposure CD8+ T cells produce IFN-g and express surface CD107a consistent with their acquisition of effector function. Therefore, this model might be useful for testing vaccines oriented towards generation of both humoral and cellular immunity. doi:10.1016/j.clim.2008.03.077 OR.71. Histoplasma capsulatum Cell Wall β-glucan Induced Lipid Body Formation and LTB4 Generation Through TLR2 And CD18 Receptors Carlos Sorgi, 1 Alexandra Medeiros, 1 Adriana Secatto, 1 Caroline Fontanari, 1 Walter Turato, 1 Caroline Bélanger, 2 Sylvie Marleau, 2 Simone Kashima, 4 Dimas Covas, 4 Patrícia Bozza, 3 Lúcia Faccioli. 11 FCFRP - Universidade de São Paulo, Ribeirão Preto, Brazil; 2 Université de Montréal, Montréal, QC, Canada; 3 Fiocruz, Rio de Janeiro, Brazil; 4 FMRP - Universidade de São Paulo, Ribeirão Preto, Brazil Histoplasma capsulatum (Hc) is a facultative, intracel- lular parasite of worldwide importance. Infection with Hc produces a broad spectrum of disease, and may progress to life-threatening systemic disease, particularly in indivi- duals with AIDS. Resolution of histoplasmosis is associated with activation of cell-mediated immunity and LTB4 play an important role in this event. Lipid bodies (LB) are specialized cytoplasmic domains for eicosanoid-forming enzyme localization. Herein, we investigated the LB for- mation in histoplasmosis and the functions that these structures could play in innate immunity. LB formation in leukocytes from C57BL/6 Hc-infected mice peaks at 2 days post infection and it correlated with the enhanced generation of both LTB4 and PGE2. Alveolar leukocytes cultured with live or dead Hc also presented an increased LB formation. The yeast alkali-insoluble fraction 1 (F1), which contains mainly β-glucan isolated from the cell wall of Hc, induced a concentrate and time-dependent increased LBs numbers, correlated with enchanted in LTB4 generation and eicosanoids 5-LO enzyme localization, demonstrating that β-glucan have a role for recognition and signaling to induce LB formation. LB induced by β- glucan in leukocytes from CD18 Low , 5-LO -/- and TLR2 -/- mice were totally inhibited. Results with PAF and BLT1 receptor antagonists showed that both lipid mediators are involved in cell signalization for LB. These results demon- strated that Hc induces LB formation and it correlated with eicosanoids production, suggesting a role for these lipid-enriched organelles in host defense during fungal infection. doi:10.1016/j.clim.2008.03.078 OR.72. Impact of Bacterial Colonization in Normal Human Livers on Activation of Toll-like Receptor 4 and Nuclear Factor kappa B and Their Possible Role in Maintaining Homeostasis S30 Abstracts
Transcript of OR.70. Humanized Mice to Test Influenza Vaccines
Cryptococcus neoformans is an opportunistic yeast thatcauses meningoencephalitis and significant mortality inpatients with impaired immune systems. Several yeast cellwall proteins are recognized by the innate immune system inmice and humans, however the molecular mechanisms andreceptors used by immune cells to bind and trigger cellactivation to C. neoformans have not been fully elucidated.Using a high-throughput RNAi screen we found that CD36, aclass B scavenger receptor, mediates macrophage activationto C. neoformans stimulation. Here we report that macro-phages isolated from CD36-/- mice had marked reduction inbinding and phagocytosis to C. neoformans and to C.neoformans-induced production of cytokines and chemo-kines in vitro. In addition, we show that CD36 collaborateswith TLR2 for C. neoformans-induced cytokine and chemo-kine production. Finally, we found that the number of yeastcells in the liver and spleen of C. neoformans infectedCD36-/- mice were significantly higher than wild-type mice.In contrast, we found that the expression of proinflammatorycytokines and chemokines in the organs of C. neoformansinfected CD36-/- mice were significantly reduced than wild-type mice. Together these data demonstrate that CD36 playsa significant role in the recognition of C. neoformans bymediating binding and proinflammatory cytokineproduction.
doi:10.1016/j.clim.2008.03.076
Fighting PathogensSunday, June 8
2:45 pm–4:45 pm
OR.70. Humanized Mice to Test Influenza VaccinesChun Yu, Mike Gallegos, Florentina Marches, SandraZurawski, Gerard Zurawski, Jacques Banchereau,A. Karolina Palucka. Baylor Institute for ImmunologyResearch, Dallas, TX
Critical to the development of novel vaccines is theavailability of in vivo models of the human immune sys-tem that permit testing of vaccine efficacy. Here, we usedNOD/SCID beta2m-/- immunodeficient mice which, whenengrafted with human CD34+ hematopoietic progenitors,develop all subsets of human dendritic cells (DCs) and B cells.T cells and their subsets can be reconstituted by adoptivetransfer. Humice can develop Influenza-specific immunityupon vaccination with ex vivo generated DCs. Vaccinationwith heat-inactivated Influenza virus pulsed DCs leads to theexpansion of FluM1 tetramer binding CD8+ T cells and thedevelopment of Influenza-specific immunoglobulins in theserum. Importantly, influenza-specific immunoglobulins areprotective and can inhibit Influenza virus-induced hemag-glutination. We also show that these mice can generaterecall CD8+ T cell responses upon exposure to seasonalinfluenza virus vaccines: live attenuated trivalent vaccine,i.e. FluMist; or killed trivalent vaccine, i.e., Fluzone. CD8+ Tcells specific to two influenza antigens FluM1 and NS1 can bedetected in the blood of mice vaccinated with FluMist butnot in mice vaccinated with Fluzone. Specific Tcells are also
present in the spleen and peripheral tissues (lung) demon-strating that human T cells can utilize murine signals fortrafficking and extravasation. Upon short-term ex vivoantigen exposure CD8+ T cells produce IFN-g and expresssurface CD107a consistent with their acquisition of effectorfunction. Therefore, this model might be useful for testingvaccines oriented towards generation of both humoral andcellular immunity.
doi:10.1016/j.clim.2008.03.077
OR.71. Histoplasma capsulatum Cell Wall β-glucanInduced Lipid Body Formation and LTB4 GenerationThrough TLR2 And CD18 ReceptorsCarlos Sorgi,1 Alexandra Medeiros,1 Adriana Secatto,1
Caroline Fontanari,1 Walter Turato,1 Caroline Bélanger,2
Sylvie Marleau,2 Simone Kashima,4 Dimas Covas,4
Patrícia Bozza,3 Lúcia Faccioli.1 1FCFRP - Universidade de SãoPaulo, Ribeirão Preto, Brazil; 2Université de Montréal,Montréal, QC, Canada; 3Fiocruz, Rio de Janeiro, Brazil;4FMRP - Universidade de São Paulo, Ribeirão Preto, Brazil
Histoplasma capsulatum (Hc) is a facultative, intracel-lular parasite of worldwide importance. Infection with Hcproduces a broad spectrum of disease, and may progress tolife-threatening systemic disease, particularly in indivi-duals with AIDS. Resolution of histoplasmosis is associatedwith activation of cell-mediated immunity and LTB4 playan important role in this event. Lipid bodies (LB) arespecialized cytoplasmic domains for eicosanoid-formingenzyme localization. Herein, we investigated the LB for-mation in histoplasmosis and the functions that thesestructures could play in innate immunity. LB formationin leukocytes from C57BL/6 Hc-infected mice peaks at2 days post infection and it correlated with the enhancedgeneration of both LTB4 and PGE2. Alveolar leukocytescultured with live or dead Hc also presented an increasedLB formation. The yeast alkali-insoluble fraction 1 (F1),which contains mainly β-glucan isolated from the cellwall of Hc, induced a concentrate and time-dependentincreased LBs numbers, correlated with enchanted in LTB4generation and eicosanoids 5-LO enzyme localization,demonstrating that β-glucan have a role for recognitionand signaling to induce LB formation. LB induced by β-glucan in leukocytes from CD18Low, 5-LO-/- and TLR2-/-
mice were totally inhibited. Results with PAF and BLT1receptor antagonists showed that both lipid mediators areinvolved in cell signalization for LB. These results demon-strated that Hc induces LB formation and it correlatedwith eicosanoids production, suggesting a role for theselipid-enriched organelles in host defense during fungalinfection.
doi:10.1016/j.clim.2008.03.078
OR.72. Impact of Bacterial Colonization in NormalHuman Livers on Activation of Toll-like Receptor 4and Nuclear Factor kappa B and Their Possible Rolein Maintaining Homeostasis
S30 Abstracts
