2 signaling pathways

Abstracts / Cytokine 48 (2009) 46–90 apoptotic response triggered by the cyclic peptide in WISH cells, supporting this chimera as a novel agent potentially useful for cancer therapy. doi:10.1016/j.cyto.2009.07.241

PP1-119 The evaluation of immunological safety of the CombiHIVvac candidate vaccine against HIV-1

73

32, 145–147 were replaced with other amino acids. After the 2nd round of panning against TNFR1, we screened 23 candidate TNFR1-selective antagonists (T11–T33) by ELISA and bioassay. One of these, T17, had an almost identical affinity for TNFR1 as T2, and inhibited TNFR1-mediated bioactivity as well as T2. On the other hand, its affinity for TNFR2 was 40-fold lower than T2. Consistent with this finding, T17 mediated lower TNFR2-mediated responses than T2. Thus, T17 possesses higher selectivity for TNFR1 binding and stimulates less TNFR2-mediated response than T2. Therefore, use of T17 might reduce the risk of side effects, such as infections, when applying TNF blockade as therapy for autoimmune diseases.

S.G. Gamaley, G.M. Sysoeva, E.D. Danilenko, V.I. Masycheva, L.I. Karpenko, Poster Presentation I The evaluation of immunologycal safety of the CombiHIVvac candidate vaccine against HIV-1 S.G. Gamaley, G.M. Sysoeva, E.D. Danilenko, V.I. Masycheva, L.I. Karpenko, FSRI State Research Centre of Virology and Biotechnology ‘‘Vector”, Rospotrebnadzor, Koltsovo, Novosibirsk Region, Russia

doi:10.1016/j.cyto.2009.07.243

The task of constructing the vaccine against HIV/AIDS is extremely urgent, which is connected with a rapid increase in the number of HIV-infected patients around the world. The difficulty of constructing the vaccine against AIDS is connected with high variability of the virus, its ability to adapt to the immune system, induce immunopathology and the lack of experimental models for evaluation of the vaccine effectiveness. One of the most promising approaches to construction of effective vaccines against HIV is connected with identification of B- and T-cellular epitopes in viral proteins and construction of synthetic polyepitope vaccines on their basis. The combined CombiHIVvac vaccine against HIV-1 containing polyepitope B- and T-cellular immunogenes TBI and TCI has been constructed by the specialists of the FSRI SRC VB ‘‘Vector”. As shown earlier CombiHIVvac induces HIVspecific immune response. The goal of the present study was to evaluate immunological safety of CombiHIVvac. Immunotoxic properties of the CombiHIVvac vaccine were studied in BALB/c mice. The weight of lymphoid organs, the number of antibody-forming spleen cells, hemolysine titers, DTH reaction and activity of peritoneal macrophages were evaluated. Allergic activity of the vaccine was evaluated by the signs of the anaphylactic response and skin sensitivity in guinea-pigs. The studies were carried out compliance with Sanitary Rules 3.3.2.561-96 (State trials and registrations of new immunobiological preparations for human medicine). It has been shown that the CombiHIVvac vaccine did not affect the weight of lymphoid organs and caused a transient increase in phagocytic activity of peritoneal macrophages, LPS-induced proliferative activity of B-lymphocytes and a decrease in the relative number of antibody-forming spleen cells. Immunization of mice with the vaccine led to a decrease in DTH reaction index which maintained for three weeks after the second immunization. As experimentally shown, the administration of the CombiHIVvac vaccine to guinea-pigs did not lead to the anaphylactic response, but caused a skin-sensibilizing effect. These data suggest that the vaccine has no marked immunotoxic properties though it should be taken into consideration that the vaccine may attenuate cellular immunity in response to foreign antigens and cause local allergic reactions.

Blanka Vicenova, Miroslava Buryskova, Ladislav Burysek, Josef Novak, Martin Pospisek, Poster Presentation I Dissecting interactions between interleukin-1alpha and histone acetyltransferase complexes by in vivo protein-tagging and immunoprecipitation techniques Blanka Vicenova 1, Miroslava Buryskova 2, Ladislav Burysek 2, Josef Novak 1, Martin Pospisek 1, 1 Laboratory of RNA Biochemistry, Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Czech Republic, 2 Protean s.r.o., Pod Lesem 9, Dobra Voda u Ceskych Budejovic, Czech Republic

PP1-121 Dissecting interactions between interleukin-1alpha and histone acetyltransferase complexes by in vivo protein-tagging and immunoprecipitation techniques

Interleukin-1alpha (IL-1a), a master proinflammatory cytokine regulator and key player in host immune responses, has been extensively studied for its ability to contribute to various autoimmune and inflammation-linked disorders. However, even if being at least in part secreted from cells and acting as a signal molecule via IL-1 receptors in the plasma membrane, a significant portion of the 31-kDa IL-1a precursor remains in the cell nucleus where it interacts with several proteins including the growth inhibitor necdin, the anti-apoptotic protein HAX-1 or elements of the RNA processing apparatus. Furthermore, nuclear IL-1a co-operates by an unknown manner with histone acetyltransferases (HATs) on transcriptional regulation of target genes. We discovered and characterized the interaction of IL-1a with HATs employing the yeast model and confirmed it further in mammalian cells. Currently we attempt to get more inside the IL-1a nuclear function by systematic gene-disruption approach combined with co-immunoprecipitation of IL-1a with selected members of yeast HAT complexes. We show that yeast model unexpectedly possesses potential to bring new information even in cytokine research and can further expand our understanding of the role of IL-1a in mammalian cell nucleus. Acknowledgements. This work was supported by Ministry of Health (Grant No. NS9819-4/2008) and by Ministry of Education, Youth and Sports (Grant Nos. MSM0021620813, MSM0021620858, LC06066). doi:10.1016/j.cyto.2009.07.244

doi:10.1016/j.cyto.2009.07.242

PP1-120 TNFR1-selectivity improvement of mutant TNF with antagonistic activity Tetsuya Nomura, Yasuhiro Abe, Masaki Inoue, Yasuo Yoshioka, Hiroyuki Kayamuro, Yohei Mukai, Madoka Taniai, Tsunetaka Ohta, Shinsaku Nakagawa, Haruhiko Kamada, Shin-ichi Tsunoda, Yasuo Tsutsumi, Poster Presentation I TNFR1-selectivity improvement of mutant TNF with antagonistic activity Tetsuya Nomura 1,2, Yasuhiro Abe 1, Masaki Inoue 1, Yasuo Yoshioka 2,3, Hiroyuki Kayamuro 1,2, Yohei Mukai 2, Madoka Taniai 4, Tsunetaka Ohta 4, Shinsaku Nakagawa 2,3, Haruhiko Kamada 1,3, Shin-ichi Tsunoda 1,2,3, Yasuo Tsutsumi 1,2,3, 1 National Institute of Biomedical Innovation (NiBio), Osaka, Japan, 2 Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, 3 The Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan, 4 Hayashibara Biochemical Laboratories, Inc., Okayama, Japan Tumor necrosis factor (TNF), which binds two types of TNF receptors (TNFR1 and TNFR2), regulates the onset and exacerbation of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In particular, TNF induces inflammatory responses predominantly through TNFR1. Therefore, we had previously identified a TNFR1-selective antagonist (T2) using a phage library expressing TNF variants in which amino acid residues at the receptor binding site were replaced with a variety of substitutions. In vivo treatment with T2 had shown that the TNFR1- blocking strategy is safe and effective for inflammatory diseases, and might overcome the risk of infections otherwise associated with TNF neutralization. In the present study, to decrease the risk of side effects when using TNF blockade, we developed a TNFR1selective antagonist with higher TNFR1-selectivity than T2. To this end, a phage display library of T2 variants was constructed, in which amino acids at positions 29, 31,

PP1-122 a-eleostearic acid induces autophagy-dependent cell death through targeting AKT/mTOR and ERK1/2 signaling pathways Jeong-Min Eom, Min-Ji Seo, Seung-Hyun Han, Chong-su Cho, Cheol-Heui Yun, Poster Presentation I a-eleostearic acid induces autophagy-dependent cell death through targeting AKT/mTOR and ERK1/2 signaling pathways Jeong-Min Eom 1, Min-Ji Seo 1, Seung-Hyun Han 2, Chong-su Cho 1, Cheol-Heui Yun 1, 1 Department of Agricultural Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea, 2 Department of Oral Microbiology & Immunology and Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea

a-Eleostearic acid (a-ESA, 9Z11E13E-18:3), a linolenic acid isomer conjugated with triene system, is a natural and biologically active compound and has been shown to possess a potent anti-tumor property. Herein, we demonstrate that a-ESA induced apoptosis and autophagy in a time- and dose-dependent manner in HeLa cells. The treatment of a-ESA to the cells caused inhibition of AKT pathway and increased subG1 area indicating induction of apoptosis. The autophagy was also induced by a-ESA treatment, which was confirmed by low levels of AKT and P70S6K pathway coincide with activation of ERK1/2 pathway and conversion from LC3 I to LC3II when compared to control. The autophagy was further tested by monodansylcadavarine (MDC) staining with fluorescence microscope and flow cytometry. It is likely that the role of autophagy is protective mechanism against cell death in a-ESA-treated HeLa cells. In fact, we found that a-ESA treatment induces reactive oxygen species (ROS), resulting in interference with AKT/mammalian target of rapamycin (mTOR) and ERK1/2 pathways. In conclusion, these results indicate that a-ESA induces apop-

74

Abstracts / Cytokine 48 (2009) 46–90

tosis in HeLa cells and at the same time the cells react to death by generation of protective autophagic and ROS molecules. doi:10.1016/j.cyto.2009.07.245

PP1-123 Both IFN-k endocytosis and the IFN-k responsive promoter activation are dependent on cholesterol Okki Cho, Seung-Ho Hong, Jungsik Kim, Sun Park, Poster Presentation I Both IFN-k endocytosis and the IFN-k responsive promoter activation are dependent on cholesterol Okki Cho, Seung-Ho Hong, Jungsik Kim, Sun Park, Department of Microbiology, Ajou University, Suwon, Southn Korea Recently, a relationship between receptor endocytosis and its signaling has been revealed for several receptors, however, the endocytosis of interferon-lambdas (IFN-k) and their receptors has not been studied. We show here that IFN-k was internalized through cholesterol-dependent, dynamin-independent and Rho family of GTPase-independent pathway in HepG2 cells. Furthermore, we demonstrate that the inhibition of IFN-k endocytosis by the cholesterol depletion suppressed the IFNk responsive promoter activation. Our results suggest the involvement of IFN-k endocytosis in its receptor signaling. doi:10.1016/j.cyto.2009.07.246

PP1-124 Antiproliferative activity of interferon-â in mucosal and cutaneous human papilloma virus –transformed keratinocytes M.V. Chiantore, S. Vannucchi, R. Accardi, M. Tommasino, E. Affabris, G. Fiorucci, G. Romeo, Poster Presentation I Antiproliferative activity of interferon-b in mucosal and cutaneous human papilloma virus – Transformed keratinocytes M.V. Chiantore 1, S. Vannucchi 1, R. Accardi 2, M. Tommasino 2, E. Affabris 3, G. Fiorucci 1,4, G. Romeo 1,5, 1 Dept of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy, 2 Infections and Cancer Biology Group, International Agency for Research on Cancer-WHO, Lyon, France, 3 Dept. of Biology, University of Rome 3, Rome, Italy, 4 Institute of Molecular Biology and Pathology, CNR, 5 Dept of Experimental Medicine, Sapienza University of Rome, Rome, Italy Human papilloma viruses (HPV) constitute a large family of viruses that can be subdivided into mucosal and cutaneous types. Mucosal high-risk HPVs are the aetiological agents of cervical cancer. Certain cutaneous HPVs, also referred as to beta types, were first isolated from patients suffering from a rare autosomal recessive cancer-prone genetic disorder, epidermodysplasia verruciformis (EV), and are consistently detected in non-melanoma skin cancer from EV, immunocompromised and normal individuals. However, a direct role of the beta HPV types in cancer development remains to be proven and the transforming properties of the majority of the cutaneous HPVs have been poorly investigated. It has been observed that the beta HPV type 38 display transforming properties that can be ascribed to E6 and E7 proteins. Recently, we have observed that IFN-b inhibits cell proliferation and deregulates cell cycle S-phase in keratinocytes transformed by both mucosal HR-HPV and cutaneous HPVs. In particular, upon longer IFN-b treatments, cutaneous HPV38 expressing cells accumulate in G1 phase and undergo senescence. IFN-b appears to induce senescence by upregulating the expression of the tumor suppressor PML. Indeed, experiments of gene silencing via specific siRNAs have shown that PML is essential in the execution of senescence program and that both p53 and pRb pathways appear to be involved. Confocal microscopy analyses to investigate co-localization in PML Nuclear bodies of specific target proteins and protein–protein interaction analyses are in progress.Moreover, with respect to the study of the anti-tumor host immune response, the natural killer cells-mediated immunosurveillance mechanism towards cells undergoing stress-induced senescent programs as well as the specific MHC class I-restricted immune response against E6 and/or E7, possibly evoked by cutaneous and mucosal HPV-transformed keratinocytes, via dendritic cells able to activate a crosspresentation process, will be reported. doi:10.1016/j.cyto.2009.07.247

PP1-125 Phagocyte-derived reactive oxygen species as suppressors of inflammation Kelly L. Brown, Karin Christenson, Martina Sundqvist, Anna Karlsson, Claes Dahlgren, Johan Bylund, Poster Presentation I

Phagocyte-derived reactive oxygen species as suppressors of inflammation Kelly L. Brown, Karin Christenson, Martina Sundqvist, Anna Karlsson, Claes Dahlgren, Johan Bylund, Department of Rheumatology and Inflammation Research, University of Gothenburg, Gothenburg, Sweden The phagocyte NADPH-oxidase is expressed by professional phagocytes and enables these cells to produce reactive oxygen species (ROS) either extracellularly or within intracellular compartments. The importance of ROS is clearly manifest in chronic granulomatous disease (CGD) patients whose phagocytes are devoid of ROS production due to mutations in genes encoding NADPH-oxidase subunits. CGD is characterized by increased susceptibility to infections as well as a variety of (sterile) inflammatory disorders indicative of an inability to properly terminate inflammatory reactions. Thus ROS production seems important both for microbial killing and for inflammatory signaling and quite likely ROS generated at different sites have different functions. Neutrophils are potently cytotoxic cells that rapidly undergo apoptosis in tissues after which they are cleared from the system by macrophages that efficiently phagocytose apoptotic cells. This clearance of senescent cells is antiinflammatory in nature and crucial for resolution of an inflammatory response. Using an in vitro system, we found gp91phox-deficient (CGD) murine macrophages to be defective regarding ingestion of apoptotic neutrophils. Human monocyte-derived macrophages in the presence of scavengers of extracellular ROS also displayed decreased phagocytosis of apoptotic neutrophils, indicating that extracellular ROS specifically facilitate this antiinflammatory event. ROS-deficient phagocytes were also inheritently hyperinflammatory and produced significantly higher levels of proinflammatory cytokines compared to WT phagocytes. Extracellular scavengers of ROS did not mediate increased production of proinflammatory cytokines from WT phagocytes, indicating that a mechanism apart from extracellular ROS was accountable. ROS have long been considered effectors of inflammatory damage, but may instead be capable of suppressing inflammatory responses and thus limit the extent of tissue damage. Our data shows that phagocyte-derived ROS act as suppressors of inflammation at several different levels and may help explain the hyperinflammatory phenotype of CGD. doi:10.1016/j.cyto.2009.07.248

PP1-126 Species-independent bioassay for quantification of antiviral type-I interferons based on luciferase-expressing Rift Valley fever virus Thomas Kuri, Matthias Habjan, Friedemann Weber, Poster Presentation I Species-independent bioassay for quantification of antiviral type-I interferons based on luciferase-expressing Rift Valley fever virus Thomas Kuri, Matthias Habjan, Friedemann Weber, IMMH (Institut fuer Medizinische Mikrobioogie, Immunologie und Hygiene), Freiburg, Germany Type-I interferons (IFN a/b ) represent an important part of the innate immune system. Quantitative measurement of IFNs in biological samples is an often needed, important issue in research, and a great variety of methods has been developed to this aim. The presence of biologically active IFN is often determined via its antiviral activity, but conventional assays are time-consuming and lack convenient readouts. An IFN-sensitive, Renilla luciferase-expressing Rift Valley Fever Virus (RVFV-Ren) was generated using reverse genetics. IFN pre-treatment of infected cells resulted in reduced Renilla luciferase expression, reflecting diminished virus replication. Human, murine and avian cells were tested for their susceptibility to RVFV-Ren after pretreatment with different amounts of species-specific IFN. RVFV-Ren was able to infect cells of all three species, and IFN-mediated inhibition of virus replication occurred in a dose dependent manner and with high sensitivity. These properties were used to develop a novel antiviral assay with a convenient luciferase-based readout in a 96well plate format. doi:10.1016/j.cyto.2009.07.249

PP1-127 A human IL10 BAC transgene reveals tissue-specific control of IL-10 expression: Implications on disease outcomes Jay H. Bream, Dilini Ranatunga, Christian M. Hedrich, Fengying Wang, Daniel W. McVicar, Nathan Nowak, Trupti Joshi, Lionel Feigenbaum, Lindsay R. Grant, Simona Stäger, Poster Presentation I A human IL10 BAC transgene reveals tissue-specific control of IL-10 expression: Implications on disease outcomes Jay H. Bream, Dilini Ranatunga, Christian M. Hedrich, Fengying Wang, Daniel W. McVicar, Nathan Nowak, Trupti Joshi, Lionel Feigenbaum, Lindsay R. Grant, Simona Stäger, Molecular Microbiology and Immunology Johns Hopkins University, Baltimore, MD Interleukin (IL)-10 is an immunoregulatory cytokine that is produced by diverse cell populations. Studies in mice suggest that the cellular source of IL-10 is thought to be a key determinant in various disease pathologies yet little is known regarding the