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PS2-055 Effect of maximal exercise on plasma concentrations interleukin-6 and related metabolic responses
Cytokine 56 (2011) 78–79
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Poster Session 2: Cytokines and Signal Transduction 1 PS2-054 Biological and structural characterization of human TNFR2-selective TNF mutants Yasuhiro Abe a, Masaki Inoue a, Takeshi Furuya a,b, Yohei Mukai a,b, Teruya Nakamura c, Yuriko Yamagata c, Hiromi Nabeshi a,b, Tomoaki Yoshikawa a,b, Yasuo Yoshioka a,d, Kazuya Nagano a, Haruhiko Kamada a,d, Yasuo Tsutsumi a,b,d, Shin-ichi Tsunoda a,b,d, a Laboratory of Biopharmaceutical Research, National Institute of Biomedical Innovation, Osaka, Japan, b Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, c Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan, d The Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan Tumor necrosis factor alpha (TNF) is one of the attractive targets for the development of anti-inflammatory and anti-tumor drugs, because it is an important mediator in the pathogenesis of several inflammatory diseases and tumor progression. Thus, there is an increasing need to understand the TNF-receptor (TNFR1 and TNFR2) biology for the development of TNFR-selective drugs. Nonetheless, the role of TNFRs, especially that of TNFR2, remains poorly understood. As part of analyzing the mechanism of the two TNFRs, we have investigated the structure-function relationship of a large number of receptor selective TNF mutants, in which each amino acid residue at positions 29, 31, 32, 145, 146 and 147 is randomly replaced with other amino acids. At first, using a unique competitive panning, we optimized our phage displaybased screening technique for isolating human TNFR2 (hTNFR2) selective TNF mutants. After 2 rounds of optimized competitive panning, we have identified several hTNFR2-selective TNF mutants with high affinity and full bioactivity via hTNFR2. One advantage of our phage-display-based technique is that it can be used to obtain the sequence information of many mutants. The hTNFR2-selective mutants were highly mutated near residue 145 and conserved near residue 30. Among these mutants, the R2-7 clone revealed very high hTNFR2-selectivity (1.8 x 105ˆ fold higher than that for the wild-type TNF). Because of its high hTNFR2-selectivity and full bioactivity, the TNF mutant R2-7 would not only help in elucidating the functional role of TNFR2 but would also help in understanding the structure-function relationship of TNF/TNFR2. To further elucidate the receptor selectivity of R2-7, X-ray crystallographic analysis, and structural simulation was used to determine a feasible basis for receptor selectivity. The complex model of R2-7/TNFR1 and R2-7/TNFR2 indicated that A145D mutation could lead to a steric hindrance disrupting the interaction between the TNFR1 and R2-7. In conclusion our phage display-based screening technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity. doi:10.1016/j.cyto.2011.07.215
PS2-055 Effect of maximal exercise on plasma concentrations interleukin-6 and related metabolic responses W. Dudzinska a, A. Lubkowska a,b, B. Dolegowska c, E. Skotnicka a, a Department of Physiology, Faculty of Natural Sciences of Szczecin University, Szczecin, Poland. Felczaka 3c, 71-412 Szczecin, Poland, b Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland. Powstancow Wielkopolskich 72, 70 111 Szczecin, Poland, c Department of Laboratory Diagnostics and Molecular Medicine, Pomeranian Medical University, Szczecin, Poland. Powstanco Wielkopolskich 72, 70-111 Szczecin, Poland Effect of maximal exercise on plasma concentrations interleukin-6 and related metabolic responses. Interleukin-6 (IL-6) is a multifunctional cytokine produced by numerous cell types including immune cells, adipose tissue, and skeletal muscle. Plasma levels of IL-6 are two- to three fold higher in patients with obesity and type 2 diabetes than in lean control subjects. This elevation is highly related to increased blood glucose, decreased glucose tolerance, and decreased insulin sensitiv-
doi:10.1016/S1043-4666(11)00447-9
ity. We investigated the effects of maximal exercise on plasma IL-6 and related metabolic responses. Twenty two healthy young males (age 19 ±3.28 yr, height 178.3 ± 5.79 cm, weight 71.0 ±6.57 kg, BMI 22.2 ±1.16 kg m-2, VO2max 54.2 ±5.47 ml kg-1 min-1) participated in this study. The examined individuals were subjected to a continuous effort test with progressively increasing intensity (up to a refusal) on a cycloergometer (Kettler X-7, Germany). The blood samples were taken from the antecubital vein immediately before, after exercise, and 30 minutes after exercise to asses plasma IL-6, insulin and glucose. After adjustment for postexercise changes in plasma volume, the date indicate a significant increase in plasma IL-6 (2.0 ±0.88 vs. 2.3 ± 1.41 pg/ml), glucose (5.0 ±1.31 vs. 6.3 ±1.29 mM), and insulin (11.9 ±7.20 vs. 17.2 ±9.82 lU/mL) concentrations and homeostasis model assessment of insulin resistance (2.4 ±1.34 vs. 5.2 ±4.82), immediately after the maximal exercise. At 30 min of recovery, all metabolic measures had returned to baseline levels. The elevation in plasma IL-6, together with increased glucose and insulin concentrations immediately after maximal exercise, may sensitize tissues for postexercise glucose uptake and glycogen restoration. doi:10.1016/j.cyto.2011.07.216
PS2-056 Investigating the biochemical properties of Interleukin 23 signaling Doreen M. Floß, Christoph Garbers, Jürgen Scheller, Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Universitätsstrasse 1, D40225 Düsseldorf, Germany The heterodimeric cytokine Interleukin 23 (IL-23) is required for the development of TH17 cells, which are characterized by their production of the pro-inflammatory cytokine IL-17. TH17 cells are necessary for the clearance of extracellular pathogens during infections, but also play a critical role in the pathogenesis of several autoimmune and inflammatory diseases in different organs. Additionally, single nucleotide polymorphisms (SNPs) within the IL-23 receptor (IL-23R) locus are associated with the development of chronic inflammatory autoimmune diseases. IL-23 and Interleukin 12 (IL-12) bridge the innate and adaptive immune system but have different functions. Both cytokines share the common p40 subunit but contain unique four-helixbundle p19 (IL-23) and p35 (IL-12) subunits. Furthermore, IL-23 and IL-12 receptor complexes consist of the shared IL-12 receptor beta 1 (IL-12Rb1) and the unique IL23R or the signal transducing IL-12 receptor chain IL-12Rb2. IL-23 signaling is mediated by the Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway including the signaling molecules Jak2, Tyk2, STAT1, STAT3, STAT4 and STAT5, and the NFjB pathway. To elucidate the IL-23 signaling pathway in vitro we generated various expression constructs with amino acid mutations and deletions within the intracellular domain (ICD) of the IL-23R. Proliferation of stable transduced Ba/F3-gp130 cells expressing the IL-12Rb1 and wild-type IL-23R or the mutated/truncated IL-23R variants has been investigated and revealed critical phosphorylation sites within the IL-23R for IL-23-induced STAT signaling. doi:10.1016/j.cyto.2011.07.217
PS2-057 Avian influenza viruses (H9N2/G1) activation of p38-MAPK and induction of tumor necrosis factor-a (TNF-a) expression are negatively regulated by protein phosphatase 2A (PP2A) Thomas YY Leon 1, Alex HM Tam 1, Davy CW Lee, Anna HY Law, Allan SY Lau, Cytokine Biology Group, Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/issn/10434666
Poster Session 2: Cytokines and Signal Transduction 1 PS2-054 Biological and structural characterization of human TNFR2-selective TNF mutants Yasuhiro Abe a, Masaki Inoue a, Takeshi Furuya a,b, Yohei Mukai a,b, Teruya Nakamura c, Yuriko Yamagata c, Hiromi Nabeshi a,b, Tomoaki Yoshikawa a,b, Yasuo Yoshioka a,d, Kazuya Nagano a, Haruhiko Kamada a,d, Yasuo Tsutsumi a,b,d, Shin-ichi Tsunoda a,b,d, a Laboratory of Biopharmaceutical Research, National Institute of Biomedical Innovation, Osaka, Japan, b Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan, c Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan, d The Center for Advanced Medical Engineering and Informatics, Osaka University, Osaka, Japan Tumor necrosis factor alpha (TNF) is one of the attractive targets for the development of anti-inflammatory and anti-tumor drugs, because it is an important mediator in the pathogenesis of several inflammatory diseases and tumor progression. Thus, there is an increasing need to understand the TNF-receptor (TNFR1 and TNFR2) biology for the development of TNFR-selective drugs. Nonetheless, the role of TNFRs, especially that of TNFR2, remains poorly understood. As part of analyzing the mechanism of the two TNFRs, we have investigated the structure-function relationship of a large number of receptor selective TNF mutants, in which each amino acid residue at positions 29, 31, 32, 145, 146 and 147 is randomly replaced with other amino acids. At first, using a unique competitive panning, we optimized our phage displaybased screening technique for isolating human TNFR2 (hTNFR2) selective TNF mutants. After 2 rounds of optimized competitive panning, we have identified several hTNFR2-selective TNF mutants with high affinity and full bioactivity via hTNFR2. One advantage of our phage-display-based technique is that it can be used to obtain the sequence information of many mutants. The hTNFR2-selective mutants were highly mutated near residue 145 and conserved near residue 30. Among these mutants, the R2-7 clone revealed very high hTNFR2-selectivity (1.8 x 105ˆ fold higher than that for the wild-type TNF). Because of its high hTNFR2-selectivity and full bioactivity, the TNF mutant R2-7 would not only help in elucidating the functional role of TNFR2 but would also help in understanding the structure-function relationship of TNF/TNFR2. To further elucidate the receptor selectivity of R2-7, X-ray crystallographic analysis, and structural simulation was used to determine a feasible basis for receptor selectivity. The complex model of R2-7/TNFR1 and R2-7/TNFR2 indicated that A145D mutation could lead to a steric hindrance disrupting the interaction between the TNFR1 and R2-7. In conclusion our phage display-based screening technique can be used to efficiently construct functional mutants for analysis of the TNF structure-function relationship, which might facilitate in silico drug design based on receptor selectivity. doi:10.1016/j.cyto.2011.07.215
PS2-055 Effect of maximal exercise on plasma concentrations interleukin-6 and related metabolic responses W. Dudzinska a, A. Lubkowska a,b, B. Dolegowska c, E. Skotnicka a, a Department of Physiology, Faculty of Natural Sciences of Szczecin University, Szczecin, Poland. Felczaka 3c, 71-412 Szczecin, Poland, b Department of Biochemistry and Medical Chemistry, Pomeranian Medical University, Szczecin, Poland. Powstancow Wielkopolskich 72, 70 111 Szczecin, Poland, c Department of Laboratory Diagnostics and Molecular Medicine, Pomeranian Medical University, Szczecin, Poland. Powstanco Wielkopolskich 72, 70-111 Szczecin, Poland Effect of maximal exercise on plasma concentrations interleukin-6 and related metabolic responses. Interleukin-6 (IL-6) is a multifunctional cytokine produced by numerous cell types including immune cells, adipose tissue, and skeletal muscle. Plasma levels of IL-6 are two- to three fold higher in patients with obesity and type 2 diabetes than in lean control subjects. This elevation is highly related to increased blood glucose, decreased glucose tolerance, and decreased insulin sensitiv-
doi:10.1016/S1043-4666(11)00447-9
ity. We investigated the effects of maximal exercise on plasma IL-6 and related metabolic responses. Twenty two healthy young males (age 19 ±3.28 yr, height 178.3 ± 5.79 cm, weight 71.0 ±6.57 kg, BMI 22.2 ±1.16 kg m-2, VO2max 54.2 ±5.47 ml kg-1 min-1) participated in this study. The examined individuals were subjected to a continuous effort test with progressively increasing intensity (up to a refusal) on a cycloergometer (Kettler X-7, Germany). The blood samples were taken from the antecubital vein immediately before, after exercise, and 30 minutes after exercise to asses plasma IL-6, insulin and glucose. After adjustment for postexercise changes in plasma volume, the date indicate a significant increase in plasma IL-6 (2.0 ±0.88 vs. 2.3 ± 1.41 pg/ml), glucose (5.0 ±1.31 vs. 6.3 ±1.29 mM), and insulin (11.9 ±7.20 vs. 17.2 ±9.82 lU/mL) concentrations and homeostasis model assessment of insulin resistance (2.4 ±1.34 vs. 5.2 ±4.82), immediately after the maximal exercise. At 30 min of recovery, all metabolic measures had returned to baseline levels. The elevation in plasma IL-6, together with increased glucose and insulin concentrations immediately after maximal exercise, may sensitize tissues for postexercise glucose uptake and glycogen restoration. doi:10.1016/j.cyto.2011.07.216
PS2-056 Investigating the biochemical properties of Interleukin 23 signaling Doreen M. Floß, Christoph Garbers, Jürgen Scheller, Institute of Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine-University, Universitätsstrasse 1, D40225 Düsseldorf, Germany The heterodimeric cytokine Interleukin 23 (IL-23) is required for the development of TH17 cells, which are characterized by their production of the pro-inflammatory cytokine IL-17. TH17 cells are necessary for the clearance of extracellular pathogens during infections, but also play a critical role in the pathogenesis of several autoimmune and inflammatory diseases in different organs. Additionally, single nucleotide polymorphisms (SNPs) within the IL-23 receptor (IL-23R) locus are associated with the development of chronic inflammatory autoimmune diseases. IL-23 and Interleukin 12 (IL-12) bridge the innate and adaptive immune system but have different functions. Both cytokines share the common p40 subunit but contain unique four-helixbundle p19 (IL-23) and p35 (IL-12) subunits. Furthermore, IL-23 and IL-12 receptor complexes consist of the shared IL-12 receptor beta 1 (IL-12Rb1) and the unique IL23R or the signal transducing IL-12 receptor chain IL-12Rb2. IL-23 signaling is mediated by the Janus kinase (Jak)-signal transducer and activator of transcription (STAT) pathway including the signaling molecules Jak2, Tyk2, STAT1, STAT3, STAT4 and STAT5, and the NFjB pathway. To elucidate the IL-23 signaling pathway in vitro we generated various expression constructs with amino acid mutations and deletions within the intracellular domain (ICD) of the IL-23R. Proliferation of stable transduced Ba/F3-gp130 cells expressing the IL-12Rb1 and wild-type IL-23R or the mutated/truncated IL-23R variants has been investigated and revealed critical phosphorylation sites within the IL-23R for IL-23-induced STAT signaling. doi:10.1016/j.cyto.2011.07.217
PS2-057 Avian influenza viruses (H9N2/G1) activation of p38-MAPK and induction of tumor necrosis factor-a (TNF-a) expression are negatively regulated by protein phosphatase 2A (PP2A) Thomas YY Leon 1, Alex HM Tam 1, Davy CW Lee, Anna HY Law, Allan SY Lau, Cytokine Biology Group, Department of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China