Demetris, AJ

Fontes, P

Fung, JJ

Kocova, M

Mullen, E

Rao, AS

Ricordi, C

Rybka, WB

Starzl, TE

Trucco, M

Ukah, FO

Vasko, C

Zeevi, A

Transplantation Proceedings

PubMed

Name not available

Human to baboon bone marrow transplantation after conditioning with non-lethal irradiation

D-Scholarship@Pitt

///1 Human-to-Baboon Bone Marrow Transplantation After Conditioning With Nonlethal Irradiation P. Fontes. A.S. Rao. C. Ricordi. A Zeevi. M. Kocova. W.B. Rybka. F.O. Ukah. E. Mullen. C. Vasko. M. Trucco. AJ. Demetris. J.J. Fung. and T.E. Starzl T HE INCREASING shortage of human donors critI-cally limits allogeneic transplantation as a treatment of choice for end-stage organ failure. As a result of this problem. there has been a resurgence of interest in xeno· transplantation. It has been universally recognized that to make transplantation across discordant species a reality. many formidable physiological barriers have to be tri-umphed. On the contrary, nonhuman primates (eg. chim-panzee. baboon. etc). by sharing many physiologic and genetic characteristics with humans. offer a more reason· able alternative as donors for xenogeneic organ transplan· tation. These observations. however. did not translate fa· vorably in clinical trials. for attempts at transplantation across concordant species were met with an undesirable outcome ,1-3 prompting many clinicians and researchers to look for or attempt to create a more suitable donor for future xenotransplantation. Our group is interested in modifying the donor's immune system prior to organ transplantation, and as such an attempt was made to create humanized baboons by infusing human bone marrow cells into a sublethally irradiated donor (baboon). We report the incidence of chimerism and in vitro immune reactivity in human-to-baboon bone mar-row chimeras. The use of such chimeric donors for future xenotransplantation is also discussed. MATERIALS AND METHODS Animals Three healthy juvenile baboons (Pappio annubLS) were obtained from the Southwestern Primate Reserve. San Antonio. Texas and were housed in an Amencan Association for Accreditation of Laboratory Animal Care (AAAlAC) approved facility at thc Montcfiore Hospital. Univer.;lty of Pittsburgh. They were rigidly screened for viral and microbial infections before and after arrival at Pittsburgh. Under light anesthesia. two animals received total lymphOid irradiation. (TI..!; 7.5 Gy) sparing the lungs. liver. head. and neck and appendages. One additional animal received total bodv irradiation (TBI: 7.5 Gy). ImmedIate I\' after irradiation. 6 x 10" unmodified cryopreserved human cadaveric bone marrow cells/kg body weight were infused into each animal through an Intravenous catheter placed in the femoral vein. and the Vital signs were mOnitored for 2 to 3 hour.; after infUSion. Human cadaveriC vertebral bodies were used as a source of bone marrow. the Isolation and cryopreservatlon of which IS described elsewhere' Each animal also received prophylactic therapy agamst Candida. herpes. and gram·negatlve infections. The survivmg animals were sacrificed ) R months after bone marrow infuSion. Posttransplant In Vitro MOnitOring: ~t~cllon of Chim~11pmK The presence of human cells In the baboon's peripheral blood mononuclear cells (PBMu) was deter· mined by flow cytometry using mouse·antihuman major hls-tocom-patibility complex (MHC) class I monoclonal antibodies (specific for the bone marrow donor). This analysis was carried out month Iv for the tir.;t 6 months and every 6 months thereafter. Probes directed against the bone marrow donor's chonogonadolrophin gene (CG) were also used to detect chimerism by polymerase chain reaction (PCR). in the baboon's PBMC every 6 months poSit ram· plant and additionally In all tissues at the lime of sacrifice. In Viero Immune Tesllng All In vitro immune testing was earned out at the time of sacrifice ( I K months posltransplant). A constant dose of PBMu obtained from the 110\iO survIVing haOoons was used as a responder in a one-way allo· and xenogeneic mixed lymphocyte reaction (MLR). For xenogeneic MLR. 'Y-irradiated cryoprescrved human PBMu (donor and third·party specific) were used. Con-trols included incubation of responders and stimulator.; in RPMI ) 640 alone and eoculture of syngeneic stimulators and responders. The cells were cultured for 5 days at 370C in 5% CO2 in air. and for the last 18 to 24 hour.; I/LCi of ["HI thymidine was added per well. The cells were harvested onto glass filtermats and radIOactive thymidine incorporation was determined by liquid scintillation counting. Cionogelllc Progl!lIlcor Cdl Assays. Bone: marrow was obtained from the baboons' vertebral bodies at the lime of sacrifice. and 2 x H}' cells were cultured in each tissue culture plate (35 mm) containing Terry Fox medium for 14 days at 37"C in 5% CO: in air. At the end of this peflod. colonies of hurst-forming-uOit ervthrOid (BFU·E). colony-forming·umt granulocyte and macrophage (CFU-GM). and colony-forming-unit granulocyte. erythrocyte. macro· phage, and megakaryocvte (CFU-GEMM) were individually scored and harvested for blind PCR detecllon of donor DNA RESULTS AND DISCUSSION Although the infusion of bone marrow was uneventful in all baboons. the animal receiving TBI (n = I) died within 14 days of irradiation due to aplastic marrow. Therefore. this From the Pittsburgh Transplantation Institute and the Depart-ment(s) of Surgery (P.F .. A.S.R .. C.V .. J.J.F .. T.E.S.). Pathology (A.S.R.. A.Z .. A.J.D.). PediatriCS (E.M.), and Medicine (W.S.R.), University of Pittsburgh Medical Center. Pittsburgh. Pennsylva-nia; The Diabetes Research Institute (C.R.). MIami, Flonda: The University of Iowa (M.K .. M.T.). Iowa City. Iowa; and Good Samaritan Hospital (F.O.U.). West ISlip. New York. Supported by NIH grant DK29961. Address repnnt requests to Thomas E. Stanl, MD. PhD. Department of Surgery. University of Pittsburgh. 3601 Fifth Avenue, 5C Falk Clinic. Pittsburgh. PA 15213. ~ 1994 by Appleton & Lange 0041-1345/94/$3.001 + 0 Transplantation Proceedings. Vol 26. No 6 (December). 1994: pp 3367-3368 3367 3368 animal will not be dl"Cussed any further. The other two animals that received sublethal TLI conditioning prior to human bone marrow transplantation survived for the dura-tion of this study Ef~ months). Donor cells were not detectable by FACScan at any time after bone marrow infusion In the PBMCs of both animals. However. PCR detection of donor-specific CG gene yielded a positive result in the PBMCs of one animal for up to 18 months after bone marrow transplantation: whereas in the other animal. donor DNA was detectable during the first 6 months. which dwindled thereafter to an undetectable level. The presence of donor cells was also analyzed by PCR in different baboon tissues which were harvested at the time of sacrifice. Donor DNA was present in the bone marrow and liver of both animals. whereas the other tissues are currently being analyzed. Clonogenic progenitor cell assays carried out on baboons' bone marrow harvested at the lime of sacrifice produced colonies of BFU, CFU-GM. and CFU-GEMM in both animals which. when tested for the pres-FONTES. RAO. RICORDI ET AL ence of donor DNA by PCR. yielded positive results. This suggests that the human hone marrow has fully engrafted. Because in vitro immune testing was only done at the time of sacrifice. it is rather difficult to extrapolate the results of this analysis. Nevertheless. both animals exhibited compar-atively similar responses against the donor and third-party stimulators as well as against allogeneic cells. The use of nonlethal irradiation followed by reconstitu-tion with recipient-type bone marrow cells might offer a reasonable alternative to modify potential donors for future xenotransplantation. REFERENCES I. Starzl TE. Marchiono n... Peters GN. et al: Transplantation 2:752. 19~ 2. Rccmtsma K. McCracken BB. Schlcgo:l JU. cl al: Ann Surg 160:384. 1964 3. Starzl TE. Fung 11. Tzakis A. et aJ: Lancel 341:65. 1993 4. Fontes PA. Rao AS. Demetris N. et al: Lancel. 344: 151. 1994 

Human-to-baboon bone marrow transplantation after conditioning with nonlethal irradiation

FONTES, P

RAO, A. S

DEMETRIS, A. J

FUNG, J. J

STARZL, T. E

RICORDI, C

ZEEVI, A

KOCOVA, M

RYBKA, W. B

UKAH, F. O

MULLEN, E

VASKO, C

TRUCCO, M

University of Miami: Scholarship Miami

http://d-scholarship.pitt.edu/5067/1/31735062124767.pdf

Human-to-baboon bone marrow transplantation after conditioning with nonlethal irradiation

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