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Abstract / Cytokine 63 (2013) 243–314 and Metabolism, Pfizer, La Jolla, CA, USA, b Experimental Medicine, Pfizer, San Francisco, CA, USA In vitro (or ex vivo) quantification of cellular parameters is often employed to inform systems models that define the relationship between drug exposure and response. We developed two flow cytometry based assays to evaluate the interaction between the IL-7 receptor (IL-7R) and an anti-IL-7R monoclonal antibody (Ab1). IL-7R occupancy and rate of internalization in the presence of Ab1 was determined using flow cytometry in monkeys and human whole blood. Two commercially available antibodies were used to detect IL-7R. One detected ‘‘free” and the other determined ‘‘total” receptor. Receptor internalization was measured ex vivo at various time points at 37 °C. The receptor occupancy assay was used in vivo following administration of Ab1 into cynomolgus monkeys at 0.3, 3, and 30 mg/kg. The amount of Ab1 bound to receptor and the total amount of receptor was quantified. The ex vivo receptor occupancy mean IC50 values in monkey and human whole blood were 5.64 ± 2.38 and 9.39 ± 1.54 ng/ml, respectively. The ex vivo rate of internalization of Ab1-IL-7R complex was characterized by a slope of 384 ± 24 and 2482 ± 319, for monkey and human, respectively. In the in vivo monkey study, no free IL-7R was detectable on the CD3 T cell surface at 0.25 h post-dose in all treatment groups. Free receptor levels returned to 78% on day 8 at 0.3 mg/kg, 55% on day 15 at 3 mg/kg, and 12% on day 15 at 30 mg/kg, compared to pre-dose levels. By Day 22, free IL-7R levels in all dose groups returned to their baseline. Ab1 treatment resulted in a significant reduction in total IL-7R. Effective biomeasures provide a link between species and the ex vivo and in vivo systems. Integration of these values into systems models reduces the number of model assumptions and increases the likelihood of success.

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Objective: Orthodontically induced inflammatory root resorption (OIIRR) is one of the most difficult to predict accidental symptom in orthodontic treatment. Orthodontic forces are known to produce mechanical damage and inflammatory reactions in the periodontal ligament (PDL). Notch signaling pathway is a highly conserved cell signaling system that plays an important role in various cell differentiation processes including bone homeostasis. However, the role of Notch signaling in tooth movement and root resorption in orthodontic treatment has not fully understood. We examined the effects of orthodontic force on Notch and Jagged1(Notch ligand) expression in human PDL (hPDL) cells in vitro, andthe expression of Jagged1, RANKL and IL-6 in root resorption area during experimental tooth movement in rats. Materials and Methods: Different compression forces (CF) were applied to hPDL cells for up to 24 h. With/ without c-secretase inhibitor (GSI; inhibitor of Notch signaling). The expression of Jagged1, RANKL and IL-6 mRNA was determined by using real-time PCR. In animal study, rats were subjected to an orthodontic force of 10 or 50 g with a closed coil spring. Experimental tooth movement was undertaken for 7 days. Each sample was prepared for HE and immunohistochemistry staining for TRAP, Jagged1, RANKL and IL-6 in root resorption area. Results: CF increased the expression of Jagged1, RANKL and IL-6mRNA from hPDL cells. Moreover, GSI suppresses CF-induced expression of these mRNA. On days 7, immunoreactivity for Jagged1, RANKL and IL-6 was detected in odontoclasts with an orthodontic force of 50 g, but not 10 g. Conclusions: These results indicate that CF may modulate the mRNA expression of RANKL and IL-6 via Notch signaling in hPDL cells. Notch signaling might be involved in the progress of inflammation in PDL tissue and the incidence of severe root resorption during orthodontic treatment. http://dx.doi.org/10.1016/j.cyto.2013.06.149

http://dx.doi.org/10.1016/j.cyto.2013.06.147

145 The erthyrocyte cytosolic 50 -nucleotidase III is a broad tissue IFN-stimulated gene that limits NF-jB-mediated inflammatory response Latifa Al-Haj, Khalid S.A. Khabar, Molecular BioMedicine Program, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia Type-I interferon (IFN) has been shown to inhibit inflammatory processes such as NF-jB activation and cytokine production through largely unknown mechanisms. We have found that pyrimidine 50 -nucleotidase (PN-I or NT5C3), previously designated as erythrocyte-specific gene product, is stimulated by IFN in a number of cell types, including normal, transformed, epithelial and fibroblast cells. Type-I IFN but not IFN-c exerts dose-dependent stimulation of NT5C3 mRNA and protein. This activation requires Stat1/tyk2 factors and ISRE binding. NT5C3 was also induced by a number of viruses and pro-inflammatory cytokines. Ectopic over-expression of NT5C3 in HEK293 cells significantly reduced TNF-a -mediated NF-jB reporter activity. IFN-a-mediated suppression of the chemokine IL-8 mRNA and protein expression in the IFN-sensitive HeLa cells is more pronounced with NT5C3 overexpression. Down-modulation of NT5C3 in HEK293 cells significantly increased both basal and TNF-a-induced NF-jB reporter activation. In order to explore further mechanisms, we investigated upstream signaling components, and showed that NT5C3 reduced IKKb-mediated activation of NF-jB reporter.- IKKb is a kinase required to phosphorylate and degrade NF-kB inhibitor (IKb). Results were further confirmed with NT5C3 silencing showing enhancement of NF-jB reporter activity in cells overexpressing wild-type IKKb. These activities were not seen in the case of IKKb (S177E) mutant experiments suggesting that NT5C3 may interfere with IKKb related activity such as its phosphorylation. Indeed, we found that NT5C3 could reduce phosphorylated levels of IKKb during TNF-a activation. Global gene expression profiling.-and further QPCR/WB confirmatory tests.- indicated that serine/threonine protein phosphatase (PPP2R1A), which dephosphorylates IKKb expression, was enhanced in NT5C3-expressing cells. IFNmediated suppression of IL-8 expression is diminished in both NT5C3- or PPP2R1Asilenced cells. Overall, these results suggest that NT5C3 is broad ISG involved in dampening inflammation through changes in PPP2R1A/IKKb/NF-jB network with reduction in pro-inflammatory cytokines highlighting a novel mode for the antiinflammatory action of Type-I IFN. http://dx.doi.org/10.1016/j.cyto.2013.06.148

146 Compression force induces inflammatory cytokines via notch signaling in periodontal ligament cells Jun Kikuta, Masaru Yamaguchi, Kunihiko Yamada, Mami Shimizu, Mari Funakoshi, Kazutaka Kasai, Department of Orthodontics, Nihon University School of Dentistry at Matsudo, Chiba, Japan

147 Proatherogenic conditions promote autoimmune Th17 responses in vivo Young Uk Kim a,b, Hoyong Lim a, Hua Sun a, Shino Hanabuchi c, Joseph M Reynolds c, Babie Teng a,b, Yeonseok Chung a,b, a Institute of Molecular Medicine, University of Texas Medical School at Houston, Houston, TX, USA, b Graduate School of Biomedical Sciences, The University of Texas Health Science Center at Houston, Houston, TX, USA, c Immunology, MD Anderson Cancer Center, Houston, TX, USA Although patients with atherosclerosis have a higher incidence of systemic autoimmune diseases, the relationship between proatherogenic factors and autoimmune T cell responses is poorly understood. Mice lacking both LDL receptor and apolipoprotein B mRNA editing enzyme (Ldlr/Apobec1/; LDb) are hyperlipidemic and prone to atherosclerosis. Here, we show that LDb mice exhibit increased IL-17 in circulation as well as in the aortic sinus area, which was attributed to a preferential enhancement of Th17 cells in the secondary lymphoid organs. In addition, the environment within LDb mice was substantially favorable for the Th17 polarization of auto-reactive CD4+ T cells during homeostatic proliferation. In vitro, the addition of oxidized LDL, but not native LDL, promoted dendritic cell-mediated Th17 polarization by triggering IL-6 and IL-1 in a MyD88-dependent fashion. Furthermore, myelin oligodendrocyte glycoprotein (MOG)-reactive CD4+ T cells expanded in the presence of oxidized LDL expressed increased levels of Th17 signature genes, and induced more profound experimental autoimmune encephalitomyelitis (EAE) when transferred into naïve mice. Our findings demonstrate that proatherogenic factors induce the polarization and functional maturation of autoimmune Th17 cells, which may be critical for the pathogenesis of atherosclerosis and other related autoimmune diseases. http://dx.doi.org/10.1016/j.cyto.2013.06.150

148 Modulation of cytokine activity through advanced polymer conjugation Peter Kirk, Murali Addepalli, Cherie Ali, Thomas Chang, Jicai Huang, Steve Lee, Paul Sims, Laurie VanderVeen, Yujun Wang, Deborah Charych, Nektar Therapeutics, San Francisco, CA, USA Cytokines have proven to be effective therapies in multiple indications including those in oncology, inflammation, and immunology. Modification of cytokines for therapeutic use by covalent attachment of a polymer such as polyethylene glycol (PEG) has been used to reduce the renal clearance rate and therefore extend exposure. The next generation of polymer conjugation technology enables more complex and profound modification of cytokine activity. With a deeper understanding of the effect polymer size and architecture have on biodistribution, coupled to the development of polymer-protein linkers with differing site-selectivity and variable release rates, the therapeutic potential of cytokines can be significantly enhanced through polymer conjugation. The engineered cytokine NKTR-214 is a multi-PEGylated form of IL-2