Carroll, P

Demetris, AJ

Fontes, P

Jordan, M

Rao, AS

Rybka, W

Scantlebury, VP

Shapiro, R

Starzl, TE

Trucco, M

Vivas, C

Zeevi, A

English

PubMed

D-Scholarship@Pitt

- .. --~--K---------------------0041-1337/95/5902-306$03.0010 'I'RANSPLANTATIO:-; Copynght © 1995 by Williams & Wilkins Vol. 59. 306-309. No.2. Januarv n. 1995 Printed In U.S.A. Transplantation ® BRIEF COMMUNICATIONS COMBINED KIDNEY/BONE MARROW TRANSPLANTATION-EVIDENCE OF AUGMENTATION OF CHIMERISM1,2 RON SHAPIRO.3.4 ABDUL S_ RAO,3.5 PAULO FONTES,3 MARK JORDAN,3 VELMA P_ SCANTLEBURy.3 CARLOS VIVAS,3 A~qelkv J. DEMETRIS;'j ADRIANA ZEEVI,5 WITOLD RYBKA,6 PATRICIA CARROLL,6 MAsSIMO TRucco! AND THOMAS E. STARZL,3 Pittsburgh Transplantatwn Institute. Departments of Surgery, Pathology, Medicine. and Pediatrics. University of Pittsburgh Medical Center. Pittsburgh, Pennsylvania 15213 Recent studies on patients who have maintained stable hepatic and renal allograft function for 10-28 vears have revealed systemIc microchimerism in all (1. 2l. With the be-lief that this is the essential mechanism of allograft accep-tance. we sought to augment the spontaneous chimerism in newly transplanted recipients. and began a program of com-bined simultaneous kidney/bone marrow transplantation without pretransplant radiation or other cytoreduction therapy (3), Postoperative immunosuppression was with FK506 and prednisone. using these drugs in the same way as in standard practice (4). Seventeen cadaver kidney recipients were entered into the bone marrow augmentation series up to .June 1994. All are well. We report here the first 10. whose simultaneous kidney-bone marrow transplantations were between December 14. 1992 and December 14. 1993. '!\vo of them also received pan-creatic islets. The mean recipient age was 41.32: 13.7 years (range 24.6-ti3.4). All patients were undergoing their first transplantatIOn and had a panel-reactive antibodv level of less than 150. The mean number of HLA matches' and mis-matches was zAc: 1.3 (range 1-5) and 3.22: 1.8 (range 1-6\. respectively. The mean donor age was :25.02:7.5 vears. and the mean cold ischemia time was 26.52:9.4 hr. -Eight control patients undergoing their first cadaveric kid-ney transplantation but not receiving bone marrow were also studied. The most common reason for not performing the combined procedure in the control cases was refusal of the donor family to consent to vertebral body recovery. Their mean age was 44.02:11.0 years (range 29.7-59.8). The mean number of HLA matches and mismatches was 3.5:!: 1.9 (range 1-til and 2.4c:2.0 (range O-Sl. The mean donor age was 50.32: 12.4 years. and the mean cold ischemia time was 31.32:3.6 hr. Neither Irradiation nor any other kind of recipient precon-ditiOnIng was used in the bone marrow recipients. All blood , Presented at the 20th Annual Meeting of the Amencan Socletv of Transplant Surgeons. May 18-20. 1994. Chicago, IL. : This work was supported by NIH Grant DK 29961. I Department of Surgery. I ,\ddrcss repnnt requests to Ron Shapiro. M.D .. :3601 Fifth Ave .. ·lth Floor Falk Clinic. Pittsburgh. PA 15213. Department of Pathology. " Department of Medicine . . Department of Pediatncs. transfusions. if needed, were with irradiated packed red blood cells. After completion of the renal transplant proce-dure. 3 x lOH unmodified bone marrow cellslkg, isolated from the donor vertebral bodies. were infused via central line (3). Bone marrow infusion was performed at the time of closure of the transplant incision. or shortly thereafter in the recovery room. In the 2 patients who received islets. isolation was according to previously described techniques (5), The islets were infused into the portal vein. after completion of the kidney transplant. All of the protocols were approved by the Institutional Review Board of the University of Pittsburgh. Just prior to transplantation. 40 ml of blood was drawn and placed in heparinized tubes. Postoperatively, blood was drawn weekly for the first month. and monthly thereafter. for chimerism studies. which included fluorescent activated cell sorter analysis (FACS), polymerase chain reaction (PCR), and V-chromosome analysis by both competitive PCR (cPCR) and fluorescent in-situ hybridizatIOn 1 FISH) of cytospin samples (in the case of female recipients of kidneys from the male donors!. The mean follow-up for the 10 kidnev/bone marrow recipi-ents is 9.92:4.7 months: all are alive and well. and all have functioning allografts. The mean serum creatinine and BUN are 1.62:0.4 mg/dl. and 232:8 mg/dl. Five (50%) patients have been weaned off steroids. The two islet recipients remain on insulin: they both have evidence of C-peptide production 10.44 and 0.11 pmoVml). but at levels insufficient to allow for insulin independence. '!\vo (20%) patients experienced early kidney non function and required dialysis during the first postoperative week. The incidence of acute rejection was 50% 15 patients). All rejection episodes were responsive to steroids andlor an increase in the FK 506 dosage. No patient required antilymphocyte therapy. Cytomegalovirus ICMV) was seen in 2 (20'7c) patients and was treated successfully with intrave-nous gancyclovir. Graft vs. host disease 1 GVHD) was not seen any patient. The control recipients of a kidney only have been followed for 4.9::0.5 months: all are alive and have functioning al-lografts (Table 11. The mean serum creatinine and BUN are 2.4::: 1.0 mg/dl and 50::32 mgtdl. None of these patients has been weaned off prednisone as of yet-however. this is most likely related to the relatively short follow-up. '!\vo 1:25%) patients had acute tubular necrosis (ATN), although only 1 :306 Januar; 27. 1995 BRIEF COMMUNICATIONS 307 TABLE 1. Summary of clinical course Initial function Rejection CMV Kidney/bone marrow 1 ~o No No 2 Yes Yes No :3 Yes No No 4 Yes No No 5 Yes Yes Yes 6 Yes Yes No 7 Yes No Yes 8 )1'0 Yes No 9 Yes Yes No 10 Yes No No 8 (80'K) 5 (50%) 2(20%) Kidney alone 1 Yes No No 2 Yes Yes No 3 No Yes Yes 4 Yes No No 5 Yes Yes Yes 6 Yes No No 7 Yes Yes No Il No Yes No 6 I 75('<cl 5 (63'k) 2125'}) ( 13%) required dialysis during the first week after transplan-tation. The incidence of acute rejection was 63% (5 patients J. All rejections were responsive to steroids and an increase in FK506. CMV was seen in 2 (25%) patients; both patients were successfully treated with gancyclovir. GVHD was not seen. In the kidneylbone marrow patients. evidence of chimerism was present by at least one of the three modalities in 9 of 9 (100%) evaluable patients (Table 21. The tenth patient, who TABLE 2. Testing for mlcrochimerism Kidnl'vlhone marrow 1 .) :1 ·l ,S(O (1" 7 Il 9 10 Kidney alone 1 2 :l 4 .') (j 7 Il F'ACS" O.9'''r 2.3<:; NAd <0.5'7r 1.3'7c 3.0'7, 2.7'7r NA <0.5('<, NA NA ~A <.5l/c NA )l'A )l'A O'} <.5l}-PCR FISH' NA "' 1.0% NA l' 1.4'70 NA NA NA 0.2%" 0.5% 0.5% , All determInatlOns performed >90 days alter transplantatlOn. " Fluorescent In-SItu hybridizatlOn for Y·chromosome was per· tiJrmed In female recipients of male organs. Also receIved islets. , NA: test not performed because of lack of feaSIbility I perfect DR match and lack of sex dispanty or availabihtv of antibody to mIs-matched HLA A. 13 antigens). . Chimerism was also detected by compeutive PCR and was found to be O.5{"c. Serum creatInine BUN FK506 Prednisone fmg/dl) fmg/dll fmg/day) fmg/day) 1.1 19 .36 7.5 1.3 15 20 12.5 1.1 22 9 0 1.5 28 18 2.5 2.3 37 8 12.5 1.7 22 24 0 2.0 11 4 0 1.9 19 18 10 1.2 32 4 0 1.7 28 9 0 1.6=:0.4 23=:8 15=:10 4.5=:5.5 1.3 21 12 10 1.9 39 20 10 3.5 53 30 7.5 1.4 22 21 .5 4.2 96 6 25 2.2 103 4 12.5 2.4 33 Il 5 2.4 :l2 14 7.5 2.4=:2.0 .')0=:32 14.4.!:8.8 10.3=:6.8 received a I-B, 2-DR matched same-sex allograft. was not evaluable for microchimerism by any of these technologies. FACS was positive in 5 of7 patients in whom evaluation was feasible. with a range of 0.9-3.0%. PCR for HLA disparities was positive in 8 of 8 patients in whom the study could be performed (Fig. ll. In the three female recipients of kidneys from male donors. Y-chromosome detection by PCR was posi-tive in all cases and in one, competitive PCR ((j) demon-strated a concentration of 0.5% donor DNA in PBMCs at 1 year. Using FISH for V-chromosome detection, a level of 0.2-1.4% was seen (Fig. 2). In the kidney alone patients, 3 of the B patients could not be evaluated for microchimerism by any modality, because of complete DR matching and lack of donor sex disparity. In the [) evaluable patients. :3 (60'7<,) were pOSitive for microchimer-ism by peR. 1 by HLA disparIty, and 2 by V-chromosome analysis. However. FACS failed to detect microchimerism in any of 3 evaluable patients by :3 months after transplanta-tion. Donor-specific reactivity could not be accurately assessed in 3 of the ten kidney/bone marrow recipients because there 1 3 7 14 300 c- c+ FIGURE 1. Polymerase chain reaction I peR) in a kidney/bone mar· row reCipient With a dispanty for HLA-A:29. demonstrating persIs-tent chimensm. ·_----------------------------------------------308 TRANSPLANTATION Vol. 59. No.2 FIGURE 2. Fluorescent in-situ hybridization (FISH) of a cytospin preparation of PBMCs of a female recipient of a kidneylbone marrow from a male donor: orange represents X-chromosome; green repre-sents V-chromosome. was complete DR-matching and a decreased in vitro immu-nologic response both pre- and post-transplantation. In the other 7 patients studied over time, there was one example of donor-specific nonresponsiveness and 2 of decreased donor-specific responsiveness that waxed and waned (Fig. 3), The other 4 patients retained donor-specific responsiveness. Only 5 of the 8 kidney-alone recipients were evaluable. One of the 5 has some evidence of decreasing responsiveness. These results and those with 7 subsequently treated kid-ney-bone marrow recipients have shown that kidneylbone marrow transplantation is straightforward to perform and is safe. There were no examples of GVHD. Stable chimerism estimated to be WOO-fold more dense than that occurring spontaneously (3) was regularly achieved. It should be em-phasized that (as was expected) bone marrow infusion did not influence the early events after transplantation. The inci-150 • aonor-sp«ifi~ Response 6 0 Third marty-sp«iCi~ Response Q., 125 ..; ...., 0 ~ 100 ~ c 0 ., 75 ... 0 e-o 0 c 50 0:: '1:1 f-o ::t: 25 ...., o o 49 115 99 134 16% 22S 2711 316 Post Operative Day FIGURE 3. Serial MLR responses of a kidney/bone marrow recipient. dence of acute rejection, the rates of early nonfunction. and the number cytomegalovirus infections were similar to those seen in patients receiving kidneys alone. However, treatment of these early problems was straightforward. After passing through this phase of convalescence. which was equally vola-tile with or without bone marrow augmentation. the more densely chimeric cohort of kidney-marrow recipients ap-peared to be an advantaged group. There was lOOCk patient and graft survival. and early completion of steroid weaning in half the cases. The full benefit of the chimeric state is ex-pected to take one or 2 years. or longer. This trial was begun with patients undergoing primary transplantation who had low PRAs. We are now broadening our indications for kidney/ bone marrow transplantation to include candidates undergo-ing retransplantation and/or those with high PRAs in order to assess the utility of adjuvant bone marrow in a more com-plex transplant setting. Historically important clinical attempts to facilitate kidney graft acceptance have been reported using adjunctive preop-erative donor blood transfusions (7) or delayed (by 3 weeks) cryopreserved cadaveric donor bone marrow (8, 9). Unlike the premises of these earlier trials. our hypothesis was that long-term engraftment of the infused donor cells could occur without "making space" by host preconditioning, without an undue risk of GVHD, and using the same immunosuppres-sive strategy that fostered the previously unrecognized spon-taneous development of microchimerism (1-3). The observa-tions in the kidney recipients herein reported, as well as in recipients of livers and hearts (3) are confirmatory of these predictions. Acknowledgments. We thank Jareen Flohr. R.N .. B.S.N .. Loraine Kaminski. R.N .. Regina Fenton. R.N .. B.S.N .. C.C.T.C .. Deborah Good. R.N.. B.S.N.. C.C.T.C.. Holly Woods. R.N., C.C.T.C .. Sue Bauder. R.N .. Janice Zagari, R.N., B.S.N., Jennifer Ovesney, R.N .. B.S.N .. and Sharon Orlofske. R.N. for their help with patient care; .Janet Schmelzer. Jo Harnaha. and Merrit Lutz for their help with data entry and organization; Troy Seskey and Charles Vasko for processing bone marrow and blood samples for chimerism studies; Sue Lombardozzi. :\tD .. and Mary Pavlick for excellent laboratory help; David Krakoskv for his help with J..,Tt'aph and slide preparation: Kate Carr for her help with slide preparation; and Karen Toler for her help with typing the manuscript and table and slide preparation . REFERENCES 1. Starzl TE. Demetris AJ. Murase N. lldstad S. Ricordi C. Trucco M. Cell migration. chimerism. and graft acceptance. Lancet 1992; 339: 1579. 2. Starzl TE. Demetris AJ. Trucco M. et a1. Cell migration and chimerism after whole organ transplantation: the basis of graft acceptance. Hepatology 1993; 17: 1127. :1. Fontes P. Rao AS. Demetris AJ. ct a1. Augmentation with bone marrow of donor leukocyte mIgration for kidney, liver. heart. and pancreas islet transplantation. Lancet 1994; 344: 151. ·t Shapiro R. Todo S. Starzl TE. ct a1. Kidney transplantation under FK506 immunosuppression. Transplant Proc 1991; 23: 920 . ;j. Ricordi C. Lacy PE. Finkle EH. Olack BJ. Scharp DW. Auto-mated method for isolation of human pancreatic islets. Diabe-tes 1988; 37: 413. 6. Rudert WA. Kocova M. Rao AS. Trucco M. Fine quantltation of circulating donor cells in post-transplanted chimenc reCIpients by competitive PCR. Transplantation (in press). I. Salvatierra 0 Jr. Vincenti F. Amend WJ. et al. Deliberate donor--January 27. 1995 BRIEF COMMUNICATIONS 309 specific blood transfusions prior to living related renal trans-plantation: a new approach. Ann Surg 1980: 192: 543. 8. Monaco AP. Clark AW. Brown RW. Active enhancernent of a hurnan cadaver renal allograft with ALS and donor bone rnar-row: case report of an initial atternpt. Surgery 1976; 79: 384. 9. Barber WH. Mankm JA. Laskow DA. et al. Long-term results of 0041-1337/95/5902-309$03.0010 TRANSPLANTATION Copyright © 1995 by Williams & Wilkins a controlled prospective study with transfusion of donor spe-cific bone rnarrow in 57 cadaveric renal allograft recipients. Transplantation 1991; 51: 70. Received 16 June 1994. Accepted 6 September 1994. Vol. 59. 309-311. ;-';0. 2. January 27. 1995 Printed in U. S.A. INDEFINITE SURVIVAL FOLLOWING SMALL INTESTINAL TRANSPLANTATION AFTER INTRA THYMIC INJECTION OF THE DONOR WITH RECIPIENT-TYPE SPLENOCYTES IN A RAT MODELl MARTIN L. BLAKELY, DAVID SHAFFER, HIROKI OHZATO, RITA GO'ITSCHALK, WILLEM J. VAN DER WERF, AND ANTHONY P. MONAC02 Division of Organ Transplantatwn. New England Deaconess Hospital. Harvard Medical School. Due to multiple toxicities associated with chronic nonspe-cific immunosuppressive therapy, induction of donor-specific unresponsiveness or immune tolerance has long been a goal of the transplant community (1 ). Various tolerance induction regimens have been devised that administer donor-type an-tigen to the recipient following brief conditioning in which peripheral. immunocompetent lymphoid cells are depleted or rendered nonfunctional. Immunologic tolerance induction following the intrathymic (i.t.)* injection of a specific antigen has been studied extensively recently after it was demon-strated that islet allografts survive indefinitely in rodents treated with brief immunosuppression following the i.t. in-jection of islets from the same strain (21. The phenomenon of immune tolerance following i.t_ injection of an antigen was actually reported much earlier in several different models (3, -/), The Immune unresponsiveness induced by i.t. injection of an antigen has been shown to be donor-specific but not tissue-specific-i.e., i.t. injection of various donor-type cells can induce unresponsiveness to varying tissue or organ al-lografts (5, 6). Despite intense investigation into the mechanisms in-volved in tolerance induction following i.t. injection of an antigen. several important questions remain to be answered. The optimal source of antigen, the optimal timing of admin-istration and the optimal immunosuppressive regimen. if any. are all currently unknown and probably vary depending on the model studied. The need for the continued presence and influence of the thymus with injected antigen is also \ Presented at the 20th Annual Meeting of the Arnencan Society of Transplant Surgeons. :-'Iay 18-20. 1994, Chicago, lL. , Address correspondence to Anthony P. Monaco. M.D .. New Eng-land Deaconess Hospital. CRI-213B, 194 Pilgnrn Rd .. Boston. MA 02215. • AbbreVlations: ALS. antilymphocyte serum; GVHD. graft-ver-sus-host disease: i.p. intraperitoneal: i.t. intrathymlc: i.v. intrave-nous; LBNF, (Lewis x Brown Norway I F\ hybrid: MST. rnedian sUlVlval tirne; ::)BT. small bowel transplantation. Boston. Massachusetts 02215 unknown, and with currently available transplantation mod-els it is difficult to assess. This report describes a new model in which we examine tolerance induction following i.t. injection of an antigen with the goal of providing new insights into the basic mechanisms involved in this type tolerance. We viewed the phenomenon of i.t. tolerance induction in a reciprocal fashion with respect to the current rejection models following transplantation. Our studies show that i.t. injection of recipient-type antigen into the donor following a single dose of antilymphocyte serum (ALS), in a unidirectional graft-versus-host disease (GVHDl model of small bowel transplantation (SBT) in the rat. results in specific unresponsiveness of donor-type lymphocytes against recipient-type antigen. This unresponsiveness is demonstrated by complete elimination of GVHD. which al-lows indefinite survival of untreated recipients of small bowel allografts in this model. Lewis rats. either pretreated or naive. served as small bowel donors and (Lewis x Brown Norway) F\ hybrid (LBNF1) rats were used as recipients. This model ofSBT is a unidirectional model in which only GVHD occurs (7). In an attempt to "tolerize" the donor lymphocytes against recipi-ent-type antigen the donors were treated prior to graft har-vest and transplantation. The six treatment groups are sum-marized in Table l. Rabbit antirat ALS was given at a dose of 1 ml by intraperitoneal injection on day -14 relative to graft harvest and SBT on day O. On day -9. Lewis donors were briven splenocytes (se) from different rat strains (depending on the experimental group) by direct i.t. injection of 50x 10" cells in 0.1 ml RPMI medium; each thymiC lobe received half the total dose. Following this donor pretreatment regimen. Lewis small bowel grafts were transplanted heterotopically into untreated LBNF t recipients as previously described (8), Recipients were weighed daily and assessed for clinical signs of GVHD. At the time of death, transplanted intestine. native small intestine. spleen. and skin biopsies were examined by routine histology to confirm the diagnosis of GVHD. Statis-tical analysis of survival data was performed by the Kaplan-

Combined kidney/bone marrow transplantation — Evidence of augmentation of chimerism

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Combined kidney/bone marrow transplantation - evidence for augmentation of chimerism

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Combined kidney/bone marrow transplantation — Evidence of augmentation of chimerism

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