Hyperimmune blood (Table 1) Hamster-to-rat (Table 2) anti-hamster antibody anti-idiotypic antibody Hyperimmune Hepatocyte transfection to donor antige

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2 Hyperimmune blood (Table 1) Hamster-to-rat (Table 2) anti-hamster antibody anti-idiotypic antibody Hyperimmune Hepatocyte transfection to donor antigen (Table 3) CsA mg/kg/day CsA 10 mg/kg/day (Table 3)

3 Table 1. BN Cardiac Allograft Survival in SD Rats Group Treatment (n) MST(day) 1 None 9 8.2±1.4 2 DST 9 6.2±1.3 * 3 Hyperimmune blood ±1.7 * (>3-week interval) 4 Hyperimmune blood 8 3 hours ~ 3 days (<2-week interval) 5 + Hyperimmune blood (>3-week interval) ±1.1 No immunosuppression for all rats * P<.01 compared with untreated controls MST = mean survival time Table 2. Hamster Cardiac Xenograft Survival in SD Rats Group Treatment (n) MST(day) 1 None 9 3.1±0.2 2 Hyperimmune blood ±0.3 (>2-week interval) 3 Hyperimmune blood (<2-week interval) 8 1.2±0.2 2

4 Table 3. Wistar Cardiac Allograft Survival in SD Rats Group Treatment (n) MST(day) Hyperimmune blood CsA (mg/kg/day) 1 <2-week interval >2-week interval <2-week interval >2-week interval <2-week interval >2-week interval <2-week interval >2-week interval >200 3

5 1 Sprague Dawley SD Wistar 2 Wistar SD ( 2 ) Hyperimmune hepatocyte SD ) 1 Pentobarbital 3-5mg/Kg 2 Wistar heparin 500units Nylon 8 0 Nylon 8 9 4

6 10 4) 1 pentobarbital sodium (3-5mg/Kg) 2 75% 3 10 ml (18G ) 4 portal vein 0.05% EDTA-PBS ( 50 ml) 5 10ml 0.05% collagenase-hbss solution ( 2% FBS) portal vein 6 liver 0.05% collagenase-hbss solution ( 2% FBS) 7 Petri dish 0.05% collagenase-hbss solution ( 2% FBS) 10 ml 37 o C 20 min 8 40 mesh filter 9 10% FBS-RPMI (1000 rpm 5 min) ml 10% FBS-RPMI :1 60% Percoll ( ) 20 o C 2000 rpm 20 min % FBS-RPMI % FBS-RPMI % Eosin Y 15 5) 5

7 1 Wistar-to-SD Hamster-to- SD 2 60% Percoll hepatocyte 10% FBS-RPMI ml 6) donor SD, 30 2ml 7) Mixed lymphocytes reaction (MLR) Wistar Rat and SD Rat Spleen 1 Wistar SD 10% FBS RPMI1640 plate spleen mesh transfer tube rpm 4 10min 1 3 RBC lysis buffer 10ml RBC lysis 10% FBS-RPMI rpm 4 10min 2 5 cell 6 Wistar cell 5X10 5 /100µl SD cell 5X10 5 /100µl 7 Wistar cell irradiation 3000rad 8 Wistar cell stimulator 9 Wistar cell SD cell culture 96 well microtiter plate 1% 5% 10% SD Serum total volume 200µl 10 well 3 11 Culture 5 1µ Ci 3 H-thymidine well 12 After 24hr cell harvest 13 Filter count 6

8 8) FACScan 1 RAT Spleen cell 2 cell count 3 cell /ml cell 5 medium 10ml FACScan medium 6 medium medium mix tube cell cell/tube 7 antibody 45min 8 2ml FACScan medium 9 tube 500 l FACScan medium 10 run flow 9) Antidonor Antibody Wistar Rat Spleen 1 wistar spleen 0.3%BSA-RPMI1640 medium 2 SD Serum (56 30min inactive) RPMI1640 diluted 3 Wistar spleen mesh rpm 4 10min 2 5 RBC lysis buffer RBC lysis 6 Wistar cell /ml 7 1 l Wistar cell 1 l diluted serum 8 incubated 30min 9 5 l baby rabbit complement 1 5 dilution 7

9 10 reincubated 60min 11 trypan blue 12 cell lysis lymphocytotoxic antibody titer 13 8

10 1. CsA 10 mg/kg/day 10 mg/kg/day CsA Wistar(B Cell,Stim,2000R)+B1-1W SD(T Cell, Resp)+B1-1W SD Serum cpm % 2% 1% 0.5% 0.25% 0% % Serum %SERUM 9

11 Normal wistar cell+b1-2w sd cell +B1-2w serum(enrich) 10

12 Normal wistar +B1-3w sd +B1-3w sd serum(enrich) 11

13 Normal wistar +B1-4w sd +B1-4w sd serum(enrich) 12

14 Normal wistar +B1-5w sd +B1-5w sd serum(enrich) 13

15 Normal wistar +B1-6w sd +B1-6w sd serum(enrich) 14

16 Normal wistar +B1-7w sd +B1-7w sd serum(enrich) 15

17 Normal wistar +B1-8w sd +B1-8w sd serum(enrich) 16

18 B1 cpm 17

19 (1) SD Wistar CsA Dose mg/kg/day A B C D SD Wistar Hepatocytes Wistar CsA 2.5 mg/kg/day 5 mg/kg/day 10 mg/kg/day 10 mg/kg/day 18

20 Allograft Cardiac Graft in Rats Model Wister SD Hepatocytes injection (portal vein) Donor Hepatocytes Rejection Hepatocytes (C) Rejection Hepatocytes Injection to SD(D) Injection to SD (A) Injection to SD (B) After 1 week CsA 0, 2.5, 5, 10 mg/kg/day After 2 weeks, heart of Wister transplanted to I. CsA 0, 2.5, 5, 10 mg/kg/day After 2, 3, 4 weeks, heart of Wister transplanted to SD rats II. CsA 10mg/kg/day After 5, 6, 7, 8 weeks, heart of Wister transplanted to SD Rejection Hepatocytes Injection to SD CsA 0, 2.5, 5, 10 mg/kg/day Hepatocytes of SD rats injection to Wistar rats( ) CsA 10 mg/kg/day After 4, 5, 6, 7, 8 weeks, heart of Wister transplanted to SD rats 19

21 Allograft Cardiac Graft in Rats Model Wister to SD 90/11/26 CsA Dose A Donor B Rejection Hepatocytes Injection to SD C (mg/kg/day) Hepatocytes Injection to SD 2 weeks 2 weeks 3 weeks 4 weeks 5 weeks 6 weeks 7 weeks 8 weeks D Hepatocytes

22 (II) SD Wistar Borwn Norway Hamster CsA Dose 10 mg/kg/day A B1 B2 B3 C1 C2 E F 21

23 Allograft Cardiac Graft in Rats Model (C) SD Wister Portal Vein Injection Rejection Hepatocytes Injection to Wistar (2 and 2 ) Donor Heart Transplantation SD CsA 0 10 mg/kg/day Heart Wistar Wistar Donor 22

24 Control W SD A Donor (Wistar) Hepatocytes Injection to SD B Rejection (Wistar) Hepatocytes Injection to SD Control BN SD Allograft Cardiac Graft in Rat Model Wistar SD CsA 2W 3W 4W 5W 6W 7W 8W 8W BN SD E Rejection (Wistar) Hepatocytes Injection to SD 100 2/ E-6 2. E-2 E-3 B 3. Check Wistar SD 4. 23

25 A 1. A-T 2. Donor Wistar Recipient SD 3. SD Normal Donor Hepatocytes T Wistar CsA 4. i. Normal Donor Hepatocytes T ii. B Recipient Liver Cells Normal Rejection Hepatocytes 5. Normal Donor Hepatocytes SD Normal Donor Hepatocytes Rejection Hepatocytes Rejection Hepatocytes Normal Donor Hepatocytes 6. Normal Donor Hepatocytes Rejection Hepatocytes B1 1. B1-T 2. Donor Wistar Recipient SD 3. Wistar SD SD (T) MLR Antibody Flow 4. Rejection Hepatocytes SD 5. 24

26 B2 1. B2-T 2. Donor Wistar Recipient SD 3. Wistar SD SD T Wistar CsA mg/kg/day CsA Rejection Hepatocytes mg/kg/day CsA 100 B3 1. B3-T-t 2. Donor Wistar Recipient SD 3. Wistar SD SD T Wistar CsA t MLR Antibody Flow 4. CsA Rejection Hepatocytes T t SD 5. B3-2-t B3-3-t B3-4-t 25

27 C 1. C 2. SD Wistar 3. SD Wistar Wistar Donor SD MLR Antibody Flow 4. Rejection Hepatocytes Wistar 5. i. ii. Wistar 6. PCR D 1. D-T 2. Wistar SD Brown Norway 3. Wistar SD SD (T) Brown Norway CsA 4. Rejection Hepatocytes Brown Norway 26

28 E 5. E-T 6. Wistar SD Hamster 7. Wistar SD SD (T) Hamster CsA 8. Rejection Hepatocytes Hamster SD SD SD SD SD SD SD SD SD Hepatocytes Hepatocytes Hepatocytes Hepatocytes Hepatocytes 27

29 1. MLR FACScan Antidonor Antibody Flow 2. (Hyporesponsiveness) 3. 28

30 /2/23

31 90/12/21

B99017024 00 0 -------------------------------------------------------------------------------------------------- 1 --------------------------------------------------------------------------------------------------

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