Demetris, AJ

Fung, JJ

Starzl, TE

Valdivia, LA

English

PubMed

Name not available

Differential survival of hamster-to-rat liver and cardiac xenografts under FK506 immunosuppression

D-Scholarship@Pitt

------~-----~-- -~-~ -Differential Survival of Hamster-to-Rat Liver and Cardiac Xenografts Under FK 506 Immunosuppression LA Valdivia. J.J. Fung, AJ. Demetns. and T.E. Starzl THE ~aj~r bame~ in clinical transplantation has been the hmltatlon ot donor organs. I Xenotransplantation has been considered an alternative source of organs, however strong immunolOgiC barriers are thought to make this option impractical. The immunosuppressant FK 506 has been shown to be several hundred times more potent than cyclosporine (CyA)2.) and it is capable of suppressing antibody produc· tion. 4 These qualities would justify a tnal of this drug in experimental organ xenografts. Indeed. Nakajima et als failed to prolong hamster cardiac xenogralts in rat recipi--. ents treated with FK 506. However. It has been demon-strated that untreated livers survive longer than the hearts in the same xenograft combination.6 •7 Thus. in this study we investigated the effect of FK S06 in the difficult ham· ster-to-rat liver and cardiac xenotransplant model and compared the results with those of Cy A. MATERIALS AND METHODS Male LVG hamsters 4100 to 150 g) and male Lewis rats 1240 to 280 g) served as donors and reCipients. respecuvely. OrthotopIC liver transplantation tOL TI was perfonned accordlOg to the cuI! tech-niqueK~ Donor cholecystectomy was carned out at the time of grafting and the preparation lacked anastomosIS of the hepauc artery. HeterotopIc heart transplantation tHll was perfonned by a microsurl!lcal technIque as described by Ono and Lindsey! Treatment groups were given either FK 506 or eyA by 1M IOjectlon for 14 days tshon-tenn Immunosuppression I. or FK·S06 I or 2 m!!/kgld for the first posttransplant month followed by dose reduction to O.S mgtkg every other day up to day 100 (Iong-tenn immunosuppressIon). Graft SUrvival and liver funclIon were mono ilored. Anuhamster cytotoxIc antibody levels were detennlOed by complement-dependent cytotoxicity assay (COC) as preVIously described.' BiOPSIes. postmortem specimens. or those taken at ~acrifice were fixed in 10% formalin and stained with hematoxylin and eosin. RESULTS Xenograft survival is shown in Table I. Liver xenograft recipients given low-dose FK 506 (0.25 mgt kg) rejected their grafts in 7 days, as controls. Short-term treatment With a dose of 0.5 mgtkgld FK 506 increased hepatic xenograft survival to a mean of 47 days. On the other hand. a high dose of Cy A resulted in moderate prolongation to 13 days. with five out of seven rats rejecting while sull under treatment. Long-term Immunosuppression with 1 mg/kgld FK 506 for 30 days. followed by 0.5 mgtkg every other day up to day 100. kept five out of eight recipients alive for more than 80 days after grafting. The same long-term protocol with 2 mg/kgld FK 506 prolonged liver xenograft survival for more than 100 days in 8 out of 12 rats. At the time of writing five recipients are still alive. the longest survivor alive at 200 days after transplantation and 100 days after FK 506 withdrawal. Unlike liver xenografts neither FK 506 nor CyA was able to produce clinically significant survival of HTx. Figure 1 shows the results of From the Departments of Surgery and Pathology. Umverslty of Pittsburgh. Pittsburgh. PA. Address repnnt requests to T.E. Starzl. MD. PhD. Department of Surgery. 3601 Fifth Ave. Falk Clinic 5C. PittSburgh. PA 15213. ~ 1991 by Appleton & Lange 0041·13451911$3.00/+0 Table 1. U.,.. and Cardiac Xenogl'lltt SurvIVIIl Under FK 508 or CyA Immunosupprwalon No. Organ Grouo TreatrnenI T,.,..,.. 1 None BOLT 2 FK 506: 0.25 mgtkgtd (short·tennl SOLT 3 FK 506: 0.5 mgtkgtd (shott·tennl SOLT 4 eVA: 30 mgtkgtd (short-term) 70lT 5 FK 506: 1 mgtkgtd (Iong-tenn) 80LT 6 FK 506: 2 mgtkgtd (lang-term) 120lT 7 None 6HT 8 FK 506: M~R mgtkgtd 6HT 9 FK 506: 1.0 mgtkgtd 6HT 10 FK 506: 1.5 mgtkgtd SHT 11 FK 506: 2.0 mgtkgtd SHT ~: Oava'" ~ ora:.IIlIIIiaIy CIOSInII:8Dn. .eornc-.o WIllI QI'OUII 1. 'PneumonIa ~ 10 ~ 'R ...... nil ... at 1IIe _ 01 WI'IIIntJ. ~eornc-Ko WIllI QI'OUII 1. SUIVMII Dava 6.7.7.7.7.7.7.B 7,7.7.B.8 (20).43.51.58.64 7,10.11.12.12.21.22 (12). (20). 27'. >80'. >80'. >80'. >80', >80' (23).(27). (37).50'. >100. >100. >100. >100 x S' 3.3.3.3.3.3 3.3.3.3.3.3 3.3.3.3.3.4 3.3.3.3.4 4 .......... 5.5 qr~tfln PrOClledlrIgS. '10123. No 6 (Dec:emoer), 1991: pp 3269-3271 M .... SuMY8l nme4dava) 7.0:!; 0.5 7.4:!; 0.5 47.2:!; 17.0 13.5:!; 5.S >80 >100 P~M 3.0 3.1 !: 0.4 3.2 !: 0.4 4.3 = 0.5 p NS ...:.01' "'::'.05-NS NS NS <.01' 3269 3270 GOT 2DIID lUlL 1500 10lI0 -AP 500 lUlL TP gJdL 7 5 4 - UntratId ---- CyA, 30 mglkgJd. - FK SOl 0.5 mglkgld. '0-2+-__ -? __ ~~--~--~--~ a I 12 11 24 30 Poat-traMplantllUGn daytl Fig 1. BiochemlC8l profiles in liver xenograft reapients under short-term FK 506 or CyA immunosuppressIOn. liver functions in xenograft recipients under short-term immunosuppression. FK S06 kept glutarmc oxaloacetic transaminase tGOT). alkaline phosphatase. total bilirubin. and total protem at normal or near normal levels when compared with those of controls or CyA-treated animals. The best mdicator of rejection seems to be alkaline phos-phatase. showing increasing values after FK 506 adminis-tration was stopped on day 14. The antibody response to liver xenografts on short-term immunosuppression is shown in Fig 2. After an initial rise in cytotoxic antibody titers that was similar to that of Cy A-treated recipients. FK S06-treated animals showed suppression of antibody formation. After FK S06 with-drawal on day 14. anubody titers started to rise again by the third week. Untreated liver xenograft recipients showed a rapid elevation in antibody titers from I: 1 on day o to around I :8.192 at rejection time. Long-term treatment with either I or 2 mgtkg FK 506 (Fig 3) completely suppressed the initial humoral response by the third post-transplant week. This antibody suppression was long-lasting since rats surviving for more than 100 days do not have any cytotoxIc antibody titer despite no longer receiv-ing FK 506. There was no suppression of anubody re-sponse to cardiac xenografts by either low- or high-dose FK 506. ---------------------.. -VALDIVIA. FUNG. DEMETRIS ET AL - UntrutH 81. - CyA,3Omglkg Z(MI - FK-I080.5 mglkg 512 121 32 8 2 [-] II I I 0 6 12 18 24 30 Post-transplantation days Fig 2. Antihamster cytotoxic antibody titers In liver xenograft reaptents under shOrt·term FK 506 or CyA immunosuppreSSIOn. After 30 minutes Incubation of the recipient'S serum and comple-ment With hamster lymph node cells (5 x 1 06!mL), the percentage of cells stained with trypan blue was calculated. A score of 200/0 cell death was constdered positive. Guinea pig serum diluted 1 :10 served as the source of complement. Histologic examination of liver xenografts in untreated controls at rejection time showed prominent portal and central venulitis with marked portal edema and moderate infiltrate of histiocytes. plasma cells. and lymphocytes (in order of predominance I. CyA treatment did not change these findings. One liver xenograft who received low-dose FK 506 for 2 weeks and was killed on the last day of treatment presented focal mild portal infiltrate with blastic cells. Essentially. the liver was normal except for focal rejection infiltrate. Open liver biopSies (day 24) were performed in two xenograft recipients under long-term immunosuppression with FK 506 (I and 2 mgJkg). Except .. G) ~ ,Q < -; CJ e Q. U Q) a: 81. 2CMI 512 128 32 8 2 (-I • 0 1 mglkg 2 mgJkg 6 12 18 24 30 Post-traup18ntatlon days Fig 3. Antthamster cytOtoXic antibody liters In liver xenograft r8Ql)ler1ta under tong-term FK 506 ~KFK 506 THERAPY ON LIVER. HEART XENOGRAFTS for mild bile duct proliferation there was no acute cellular rejection. The same features were observed in one rat that died from aspiration pneumonia 50 days after transplanta-tion. and in another recipient that died on day 109 of a cardiac condition due to widespread calcification oi the left atrium. One liver xenograft reCipient underwent liver bi-opsy on day 150. This animal had no antibody titers in the serum. however. histologic findings revealed that a brisk cellular acute rejection is ongoing, with biliary duct dam-age but no lobular necrosIs. This particular recipient is still alive at 200 days after transplantation and it is doing clinically well. Untreated cardiac xenografts presented characteristic humoral-mediated rejection and these findings could not be changed by FK 506. DISCUSSION -The first attempts to prevent hepatic xenograft relectlon in the hamster-to-rat model using CyA combined with sple-nectomv. ~ or the addition of total lymphOid irradiation <ILl) to the former therapy. 10 resulted in only a moderate prolongation but not in long-term recipient survival. The difficulty in this xenograft model is the fact that antlbody-complement mechanisms are primarily responsible for rejection of extrahepatic xenografts."· 12 while liver xe-nografts undergo a combination of both humoral and cellular rejection. ~ In this context. our results with FK 506 alone producing long-term survival of liver xenografts. even with clinically applicable dosing, are remarkable. Notably. we have demonstrated that only the liver is favored by this immunosuppressant because the heart succumbs to the early vigorous humoral rejection. An interesting development would be to investigate if a liver xenograft could protect the heart or other organs from rejection. as It has been demonstrated with liver allografts protectlng kidneys irom the same donors In presensltlzed humans. 13 There are several important observations to be made in this study. Naive Lewis rats do have preformed anubodies against hamster lymphocytes. though in very low titers. Because of this they would fail to Induce hyperacute rejection. In a matter of days. however. cytotoxic antibod-ies are induced in extremely high titers. Although both FK 506 and Cy A suppressed moderately this initial humoral response. antibody titers were still above the level that is usually found at the time of cardiac xenograft rejection in this model. Thus. not only hepatic allografts 14 but hepatic xenografts are unusually resistant to humoral injury. On the other hand. we have found that CyA-treated animals presented more cellular infiltrate than untreated xe-nografts. while in the recipients receiving FK 506 there 3271 were only minimal portal fntlammatory cells. Thus. the difference between both drugs might be in their ability to suppress cellular-mediated rejection. A striking finding in this study is that under FK 506 treatment the humoral and cellular responses from the .xenograft recipient waned by the end of the third week. provided that the treatment was continued. No animal presented cellular rejection or cytotoxic antibody titers while under long-term immunosuppression. no matter that the FK 506 dose was reduced. Only 50 days after FK 506 administration was stopped. cellular but not humoral re-jection is under way. This lack of antibodies would ex-plain. in part. how a xenograft recipient does not develop immune complex-related diseases. such as serum sickness and arthritis.7 In conclusion. this is the first report of a successful liver .xenograft in a model that is immunologically difficult to overcome. FK 506 is largely more etfective than eyA in this xenograft system but ItS etfect favors only the liver and not the hean xenotransplants. A successful clinical liver \enotransplantation with FK 506 immunosuppression looks highly possible. especially in a combination in which preformed antibodies are present in low titers and there is no hyperacute rejection. but in the end rejection is mainly humoral-mediated. in other words. the baboon-to-human system. REFERENCES 1. Rapaport FT. Analse D: Transplant Proc 23:899. 1990 1. Oehiai T. Sakamoto K. Nagata M. et al: Transplant Proc ~l:OM9K 1988 (suppl Il 3. Zeevi A. Duquesnov RJ. Eiras G. el al: Surg Res Commun 1:315.1987 4. Thomson AW: Transplant Proe 22:100.1990 (suppl11 5. Nakajima K. Sakamoto K. Oehiai T. et al: Transplantallon -15: 1146. 1988 6. Valdivia LA. Monden M. Gotoh M. et al: Transplant Proc 19: \lS8. 1987 7. Monden M. Valdivia LA. Gotoh M. et al: Transplantation 43:745. 1987 8. Valdivia LA. Monden M. Gotoh M. et al: Transplantation 44:759. 1987 9. Ono K. Lindsey ES: J Thorae Cardiovase Surg RT:O~K 1969 10. Yamaguchi Y. Halperin EC. Harland RC. et al: Transplan-tallon 49:13.1990 II. Van den Bogaerde J. Aspinall R. Wang MW. et al: Trans-plantation 52:15. 1991 12. Valdivia LA. Monden M. Gotoh M. et al: Transplantallon 50: 132. 1990 13. Fung JJ. Makowka L. Tzakis A. et al: Transplant Proc 20:88. 1988 (suppl I) 14. Stan! TE. lwatsuki S. Van Thiel DH. et al: Hepatology 2:614. 1982 

Differential survival of hamster-to-rat liver and cardiac xenografts under FK 506 immunosuppression

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Differential survival of hamster-to-rat liver and cardiac xenografts under FK 506 immunosuppression

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