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1 第 1 回病態制御科学系シンポジウム抄録集炎症感染とその制御話題提供者病態機構学講座 ( 世話人 : 岩月, 高柴 ) 松川昭博 ( 免疫病理学 ) 松浦栄次 ( 細胞化学 ) 大野貴司 ( 皮膚科学 ) 高柴正悟 ( 歯周病態学 ) 癌体質浸潤と新しい治療戦略話題提供者腫瘍制御学講座 ( 世話人 : 中山, 佐々木 ) 清水憲二 ( 分子遺伝学 ) 志茂剛 ( 口腔顎顔面外科学 ) 近藤英作 ( 病理学腫瘍病理 ) 渡部昌実 ( 泌尿器病態学 ) 28 年 12 月 9 日開催

3 Hepatitis C virus ConA-induced hepatitis ALT level Mice survival ConA:15 mg/kg iv ConA:2 mg/kg iv auto-antigenn Th1 Lymphocytes ALT (IU/dL) WT SOCS3Tg % survival 1 WT SOCS5Tg necrosis 1 SOCS5Tg SOCS3Tg fibrosis remodeling 5 ** ** Time (h) 5 * Time (h)

4 6 h 12 h ConA-hepatitis Hepatic apoptosis in ConA-hepatitis WT ConA:15 mg/kg iv ConA:15 mg/kg iv 6 h Liver resection SOCS3Tg SOCS5Tg WT SOCS3Tg % injured area SOCS5Tg ** ** Time (h) DNA ladder WT SOCS3Tg M nmol pna liberated per hour/g protein 1 5 Caspase activity Caspase 3 Caspase 8 Caspase ** 1 ** ** 1 WT SOCS3Tg WT SOCS3Tg WT SOCS3Tg Hepatic cytokines in ConA-hepatitis 6 h ConA:15 mg/kg iv Livers RT-PCR Cytokine levels from splenocytes ConA:15 mg/kg iv 6 h 72 h Splenocytes SUP WT SOCS3Tg IL-12 IFN CXCL9 CXCL1 TNF 14.7 ± ± 1.5* IFN 78.7 ± ± 8.7 CXCL9 8.1 ± ±.8 CXCL ± ± 14.4* CXCR ± ± 8.7** (ng/ml) 1 5 WT ** SOCS3Tg **.2 5 **.1 ** WT SOCS3Tg WT SOCS3Tg WT SOCS3Tg Hepatic lymphocytes in ConA-hepatitis Hepatic lymphocytes in ConA-hepatitis WT SOCS3Tg ConA:15 mg/kg iv 35.1 CD4 + CD69 + (%) 3 ** h FACS Splenocytes RT-PCR Relative expression 1 5 T-bet * GATA3 ConA:15 mg/kg iv NKT cells WT (%) SOCS3Tg h FACS Splenocytes RT-PCR CD8 + cells 37. (%) Relative index Perforin * WT SOCS3Tg Granzyme B CD4 CD69 WT SOCS3Tg WT SOCS3Tg WT SOCS3Tg NK1.1 CD WT SOCS3Tg CD4 CD WT SOCS3Tg 1 WT ** SOCS3Tg

5 1

10 1 NK LL-37) 28,12,9 TB elafin merged HBD-2 Huh W, Oono T et al. J Mol Med 8:22 1

Materials and Methods Total RNA from psoriatic skin One-Step RT-PCR of HBD-3 with Factor Xa recognition site in NH2 terminus Primer pair pethbd3-f 5_- ATATATGAATTCATGGGAATCATAAACACATTA pethbd3-r 5_-

11 Materials and Methods Total RNA from psoriatic skin One-Step RT-PCR of HBD-3 with Factor Xa recognition site in NH2 terminus Primer pair pethbd3-f 5_- ATATATGAATTCATGGGAATCATAAACACATTA pethbd3-r 5_- ATATATGTCGACTGTTTATTTCTTTCTTCGGCAGCA Cloned into TOPO TA cloning vector Subcloned into pet 28a vector Expression in Terrific Broth medium by induction with.2 mm IPTG Cell lysis in 6M guanidine HCl and 1 mm Tris-Cl (ph 8.) Ni-NTA resin binding Wash 4kDa Elution with 6 M guanidine HCl, 1M imidazole, and 1 mm Tris HCl (ph 6.) Transformed into BL21 codon plus cell Desalted by RP-HPLC Crude sample was collected M G S S H H H H H H S S G L V P R G S H M A S M T G G Q Q M G R G S E F I E G R 6x His-tag FXa site G I I N T L Q K Y Y C R V R G G R C A V L S C L P K E E Q I G K C S T R G R K C C R R K K HBD-3 Clevage of His-tag with Factor Xa Bactericial Assay ~ 1 6 CFU/ml of bacteria in 1 mm PBS with 1% TSB Add ~4 g/ml HBD-3 ~8 g/ml of LVFX or IPM with, 2 or 5 g/ml of HBD-3 Incubation at 37 for 9 min Question: Why does HBD-3 have potent activity against both gram(-) and (+) bacteria? Purification by RP-HPLC Plating and incubate at 37 for overnight hbd-3 GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK 16k 6k Tris-Glycine gel electrophoresis in denatured and non-reducing condition Lane 1: Marker Lane2: Crude sample of His-tag conjugated rhbd-3 Lane 3: rhbd-3 after His-tag cleavage Collection of bacteria Count colonies Pseudomonas aeruginosa Pae1 67M Leg ulcer Pae2 66F Herpetic gangrene Pae3 54M Mycosis fungoides hbd-2 GIGDPV---TCLKSGAICHPVFCPRRYKQIGTCGLPGTKCCKKP hbd-1 GLGHRSDHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK 4k MBL (+) Pseudomonas aeruginosa MBP1 67M Necrotizing fascitis MBP2 48F Fournier's gangrene MBP3 68M Burn HBD-3C-RR 2

12 stakashi@cc.okayama-u.ac.jp IgG

15 IL-6 IL-6 CRP IL-6 2 MCP-1 4,, IgG

22 1 QOL 3 1. CTGF/CCN2 CCN2 CCN2 MDA-231 CCN2 2. FAK (Thy 397) FAK FAK TAE226 TAE226

23 1 Bn Tm Tm OC 2 μm X - 2 PTHrP IL-1 IL-6 TNF- PGE2 others PTH PTH PTHrP PTHrP VD VD 3 PGE PGE 2 M-CSF RANKL OPG O R M-CSF OPG O RANK RANKL R KL ANK

24 CTGF/CCN2 CCN2 (Nakanishi et al. Endocrinology, 2) (Nishida et al. J. Cell Physiol., 2) CCN2 (Shimo T, et al., J Bone Miner Res, 25) (Shimo et al. J. Biochem., 1998, 1999) CCN2 CCN2CCN2 -/- Control IgG CCN2 Ab MDA231 WT CCN2 -/- HT18 (Shimo T, et al., Oncology 21) (Ivkovic S, et al., Development, 23)

25 CCN2 Control IgG CCN2 Ab Control IgG CCN2 Ab Tm Tm (Shimo T, et al., J Bone Miner Res, 26) CCN2 Control rccn2 PTHrP CCN2 TGF- Ca CCN2 (Shimo T, et al., Anticancer Res 28) FAKTAE226 FAK TAE226 FAK ATP FAK397 FAK Thy397 RANKL FAK FAK Ca

26 DNA RNA / in vitro, in vivo ()

29/3/26 lymphoma glioma in vivo cancer sigma-aldrich.com Molecular targeting by delivery of functional peptide/proteins via the oligopeptide-based cell permeable vector 2.

27 29/3/26 lymphoma glioma in vivo cancer sigma-aldrich.com Molecular targeting by delivery of functional peptide/proteins via the oligopeptide-based cell permeable vector 2.5nmol, p16 1nmol FITC-p16 pep. alone Wr-T + p16 pep. 2.5nmol, p16 1nmol No pep. p16 pep. alone 28% 29% protein docking domain cell permeable domain hydrophobic pocket spacer PTD G/G1: 51% intracellular uptake after 12 hrs peptide/protein transporter complex formation mix! cargo peptide/protein ( oligopeptide or protein ) cell targeting! plasma membrane cytoplasm nucleus targeting! fluorescnce intensity (fold) p16 pep. - (2 M) transporter pep-1 (2 M) Wr-T (5 M) Wr-T + p16 pep. Wr-T + p16 pep hrs phospho-prb ( Ser78 ) Actin TsA (1 ng/ml)! sigma-aldrich.com RT-PCR HeLa SP-53 BALM-14 BALM-18 K-562 YT DERL-2 BALL-1 PBMC phospho-prb expression in the p16-negative cell lines p16 INK4a Cdk4 GAPDH Western HeLa SP-53 BALM-14 BALM-18 K-562 YT DERL-2 BALL-1 PBMC phospho- Ser78 prb Leukemia / Lymphoma Actin sigma-aldrich.com 1

29/3/26 PBMC Primary lymphoma 1. x 1 5 cells 6. x 1 5 cells 2.

3. 2. 8. 4. 2. 3. 2. 2.5nmol +p161nmol 12 24 48 72 hrs 12 24 48 72 hrs 12 24 48 72 hrs 12 24 48 72 hrs cell numbers start: 2x1 5 cells x 1 5 cells 6. 5. 4. 3. 2. 12 24 48 72 hrs x 1 5 cells 15.

no peptide 1nmol viablecells%(annexin ) % 1 85.6 1 75 5 25 no peptide Transporter 43.4 p16 pep. 16. Transporter + p16 pep.

5 1 2 25 days 5 15 X Y Day 14 single administration Wr-T: 4 nmol p16 pep : 2 nmol subcutaneous inj. BL tumor (HE) 24 hrs post- injection (unfixed) sigma-aldrich.

28 29/3/26 PBMC Primary lymphoma 1. x 1 5 cells 6. x 1 5 cells 2. x 1 5 cells nmol +p161nmol hrs hrs hrs hrs cell numbers start: 2x1 5 cells x 1 5 cells hrs x 1 5 cells p16 + positive hrs incubation timehrs no peptide Wr-T alone p16 pep. alone Wr-T + p16 pep. Annexin V Trans.+p16 pep. no peptide 1nmol viablecells%(annexin ) % no peptide Transporter 43.4 p16 pep. 16. Transporter + p16 pep. 1nmol + p16 maximum diamet er of tumor (mm) control (no peptide) Transporter alone Transporter + p16 peptide days 5 15 X Y Day 14 single administration Wr-T: 4 nmol p16 pep : 2 nmol subcutaneous inj. BL tumor (HE) 24 hrs post- injection (unfixed) sigma-aldrich.com RT-PCR Wr-T p14-1c + Wr-T p16-mis + + complex phased contrast Wr-T + p14-1c Wr-T + p16-mis merge p14 p15 p16 p18 p19 GAPDH 83 bp 6 HeLa 2 p14 p16 p18 p19 p15 GAPDH 83 bp 6 U87EGFR 2 83 bp 6 Gli36EGFR 2 Cdk4 Cdk6 Hdm2 RB GAPDH He U Gli HeLa U87EGFR Gli36EGFR p14 p15 p16 p18 p19 GAPDH 83 bp 6 p14 p16 p18 p19 p15 GAPDH 2 83 bp bp hrs 48 hrs p14 pep (M) p14 pep (M) p14 ARF, p16 INK4a p53 p21 Actin p53 p21 Actin 2

29/3/26 (OD) 1.5 1..5 U87EGFR MTT Wr-T p16-mis p14-1c - - - 13 M - - - 13 M - - - 13 M 7 M 6 M - 7 M - 6 M 7 M 3 M 3 M (final conc.

(13M) Wr-T+p14-1C (7M+6M) Wr-T+p16-MIS (7M+6M) Wr-T+p14-1C+p16-MIS (7M+3M+3M) p21-s154a pep. proliferation (MTT) 1.5 1.

29 29/3/26 (OD) U87EGFR MTT Wr-T p16-mis p14-1c M M M 7 M 6 M - 7 M - 6 M 7 M 3 M 3 M (final conc.) Gli36EGFR (glioblastoma cell line) G ATGAACCTGAGGCC p53 exon7 p21 p14 HDM2 mut p53 GADD hrs Arg 248 Leu Fas Bax (%) MTT (missense mutation) 12 (OD) no treat Wr-T (13M) Wr-T+p14-1C (7M+6M) Wr-T+p16-MIS (7M+6M) Wr-T+p14-1C+p16-MIS (7M+3M+3M) p21-s154a pep. proliferation (MTT) U87EGFP U87MG U251 AO2 SK-MG-1 T98 Wr-T - 13M p16-mis p21-s154a merge cargo peptide M p16 13M 6M - 7M - 7M p21 13M 6M p16 3M + p21 3M U87EGFR 1 x1 5 cells ~ 5 days in vivo dual peptide delivery development of human glioblastoma day 2th in brain (~3mm) tumor HeLa HeLa Hs578 hepatocyte (mouse) p14 p16 p14 p16 p19 p16 Wr-T-mediated peptide delivery peptides to brain tumor.. peptide complex (Wr-T/p14-1C + Wr-T/p16-MIS complex) 1 H&E Ki-67 P <.1 Hs578 HeLa Hs578 hepatocyte GAPDH3 Rho-Dex survival rate (%) D5 D7 D9 Wr-T+ U87EGFR HeLa Hs578 hepatocyte (mouse) p14+p16 pep full (~116kDa) p16 MIS p14-1c PARP cleaved (~82kDa) (days post-implant) vehicle (n=1) Wr-T/p16-MIS + Wr- T/p14-1C (n=1) p16 MIS p14-1c merge Actin 3

Hybridization Normal human fibroblast cells mrna AAAA AAAA AAAA Reverse transcription Degradation of RNA with NaOH cdna (single strand) TTTT TTTT TTTT Immortalized human fibroblast cells

31 29/3/26 REICDkk3 NH 2 Location-11p15.1 Coiled-coil tertiary structure REIC (Reduced Expression in Immortalized Cells) 35aa, 15bp cdna Cystein-rich domain COOH REIC (Reduced Expression in Immortalized Cells) Subtractive Hybridization Normal human fibroblast cells mrna AAAA AAAA AAAA Reverse transcription Degradation of RNA with NaOH cdna (single strand) TTTT TTTT TTTT Immortalized human fibroblast cells mrna AAAA AAAA 1. 2.mRNA Signal peptide N-glycosylation site (putative) 2 ( : Biochem Biophys Res Commun 268: 2, 2) Hybridization AAAA TTTT cdna/rna hybrid TTTT non-hybridized cdna (single strand) Separation of Single strand cdna with hydroxyapatite column TTTT Cloning and Sequence 3. REIC (Tsuji et al.: Biochem Biophys Res Commun 268: 2, 2) 1

32 29/3/26 2 STAT REIC IL-4GM-CSF REIC

2004级研究生现代免疫学实验技术结业报告

2004级研究生现代免疫学实验技术结业报告 PCR PCR PCR RT-PCR PCR (Polymerase Chain Reaction,PCR) 80 DNA, DNA PCR PCR PCR DNA PCR -- -- DNA DNA 93 DNA PCR DNA DNA ( ) DNA 55 DNA DNA -- TaqDNA dntp DNA -- -- PCR PCR DNA DNA Y (1 X) n Y DNA X (Y)