Modifications of the conditioning regimen for achieving mixed chimerism and donor-specific tolerance in cynomolgus monkeys.

BACKGROUND: We demonstrated previously that a nonmyeloablative preparative regimen can induce mixed chimerism and allograft tolerance in cynomolgus monkeys. METHODS: The current studies were designed to clarify the importance and toxicity of various elements of the allotolerance conditioning regimen by: fractionating or reducing the whole-body irradiation (WBI) dosage; adding deoxyspergualine; or deleting donor bone marrow, cyclosporine, irradiation, or splenectomy. RESULTS: Monkeys treated without donor bone marrow, cyclosporine, or irradiation did not develop chimerism or long-term allograft survival. One of three monkeys treated without splenectomy developed chimerism but died of a surgical complication. The other two did not develop chimerism and rejected by day 117. Six of six monkeys treated with 300 cGy of fractionated WBI developed chimerism. Five of these recipients had long-term graft survival. Only two of four monkeys treated with 250 cGy developed chimerism, so a 2-week course of deoxyspergualine was added. This led to chimerism in two monkeys, but one died of ureteral stenosis and the other died of allograft rejection. An unanticipated high incidence of ureteral complications felt to be secondary to rejection episodes and ischemic injury was observed in the long-term surviving animals. CONCLUSIONS: All parameters of the original preparative regimen seem to be essential for consistent success. The degree of lymphocyte depletion was proportional to the WBI dose. Long-term graft survival was observed only in recipients achieving lymphocyte chimerism of > 1.5%. In this model, lymphocyte depletion seems to be the best predictor of chimerism, and significant lymphocyte chimerism seems to be important in achieving tolerance.

Tolerance in a concordant nonhuman primate model.

BACKGROUND: We have previously demonstrated that induction of mixed lymphohematopoietic chimerism resulted in donor specific renal allograft tolerance without the need for chronic immunosuppression in nonhuman primates. Here we have tested whether tolerance can be similarly induced for baboon to cynomolgus renal xenografts. METHODS: After preconditioning with anti-thymocyte globulin (ATG), nonlethal total body irradiation, and thymic irradiation, cynomolgus monkeys underwent splenectomy, native nephrectomies, and baboon marrow and renal transplants. Postoperative cyclosporine was given for 28 days. RESULTS: In Group 1 (n=2, survival= 13, 14 days), both animals developed anti-donor immunoglobulin G, had biopsy findings consistent with humoral rejection, and showed rapidly progressive xenograft failure. In Group 2 (n=5, survival=1, 16, 33, 112, 190 days), 15-deoxyspergualine was added to the regimen (Day 0-13). In one long-term survivor, donor specific hyporesponsiveness was first observed (mixed lymphocyte culture [(MLR]) on Day 48. MLR reactivity returned on Day 64 together with the development of anti-donor antibody and subsequent xenograft failure on Day 112. Donor specific T-cell hyporesponsiveness was detected in the other long-term survivor for the first 133 days, after which a donor-specific skin xenograft was placed, (survival 24 days). Following the skin graft rejection, a rise in the MLR, development of anti-donor antibody and progressive rejection of the renal xenograft were observed. CONCLUSIONS: Antibody-mediated rejection seems to constitute the major difference between concordant xenografts and allografts. Addition of 15-deoxyspergualine for 2 weeks posttransplant extended concordant primate xenograft survival to 6 months without chronic immunosuppression. In contrast to the allogeneic model, renal transplant acceptance in this xenogeneic system was interrupted by placement of a donor-specific skin graft.

Long-term outcome and alloantibody production in a non-myeloablative regimen for induction of renal allograft tolerance.

BACKGROUND: Multilineage chimerism and long-term acceptance of renal allografts has been produced in non-human primates conditioned with a nonmyeloablative regimen. Our study was undertaken to evaluate the immunological and pathological status of long-term survivors and to define the role of splenectomy and of the primarily vascularized kidney in the regimen. METHOD: Monkeys were treated with the basic regimen, including: total body irradiation, thymic irradiation, antithymocyte globulin, donor bone marrow transplantation, and a 4-week course of cyclosporine after which no further immunosuppression was given. They were divided into four groups according to the timing of kidney transplantation (KTx) and splenectomy as follows; group A (n=13): KTx and splenectomy on the day of donor bone marrow transplantation (day 0); group B (n=3): KTx on day 0 without splenectomy; group C (n=7): splenectomy on day 0 but delayed KTx until 3 to 16 weeks post-donor bone marrow transplantation; group D (n=3): both splenectomy and KTx delayed until day 120 post-donor bone marrow transplantation. RESULTS: In group A, 11 of 13 monkeys developed chimerism and 9 monkeys achieved long-term survival of 4 to 70 months without evidence of chronic vascular rejection. Alloantibodies were detected in only one long-term survivor. In contrast, all three monkeys in group B developed alloantibodies and rejected their allografts. In group C, long-term survival without alloantibody production was observed in two of three monkeys that had developed chimerism. In group D, all three recipients were sensitized and rejected the kidney allografts rapidly after transplantation. CONCLUSIONS: 1) Production of anti-donor antibody was prevented in most recipients that developed mixed chimerism in the regimens with splenectomy at the time of donor bone marrow transplantation. 2) If splenectomy is not included in the initial conditioning regimen, induction of B cell tolerance is less likely and the result is late onset of alloantibody production and allograft rejection. 3) Immediate transplantation of the kidney at the time of recipient conditioning is not essential for induction of donor specific hyporesponsiveness by bone marrow transplantation.

A study of tolerance in a concordant xenograft model.

Antibody-mediated rejection appears to constitute the major difference between concordant xenografts and allografts in nonhuman primates. Consistent with its known effect on antibody responses, 5-7 addition of DSG to the conditioning regimen has extended concordant primate xenograft survival for up to 6 months after discontinuation of conventional immunosuppression. In contrast to our observations in recipients of renal allografts, donor-specific skin graft rejection can occur and even in long-term recipients may induce rejection of a previously accepted renal xenograft.

Prolongation of cardiac xenograft survival by double filtration plasmapheresis and ex vivo immunoadsorption.

A double filtration plasmapheresis (DFPP) technique and ex vivo immunoadsorption were applied to remove natural antibodies and avoid hyperacute rejection in discordant xenotransplantation. A swine heart was heterotopically transplanted into a dog's neck after DFPP and ex vivo immunoadsorption using a swine spleen or liver. Mean graft survival time was prolonged to 240 +/- 141 min in the group treated by combined DFPP and splenic adsorption, and 225 +/- 62 min in the group treated by combined DFPP and hepatic adsorption, whereas it was 9 +/- 5 min in the group without any treatment (p < 0.01). Mean removal rates of IgG and IgM were 85.5% and 93.3%, respectively. Anti-swine lymphocytotoxic antibodies and hemagglutination antibodies were also effectively removed. Deposits of canine IgM and C3 on the vascular endothelium of the graft were observed on immunofluorescence, which suggested that natural antibodies in the IgM fraction played an important role in xenohyperacute rejection.

Effective oral administration of tacrolimus in renal transplant recipients.

BACKGROUND: Tacrolimus is usually administered orally based on mg/kg or the trough concentration (Cmin) in whole blood. However, it is not easy to determine the optimal oral dose because of considerable variation of tacrolimus bioavailability between patients. In this study, pharmacokinetics of tacrolimus in various conditioning kidney transplant recipients was investigated. METHODS: 1) Thirty-three patients were randomly assigned to participate in two groups. In 17 patients, tacrolimus was administered orally after an overnight fast with breakfast given 1 h later, and in the other 16 patients it was administered orally 1 h after breakfast. 2) In 4 patients, the same dose tacrolimus was given after an overnight fast and 0.5 h after breakfast on two consecutive days. 3) Five patients who underwent continuous ambulatory peritoneal dialysis (CAPD) before transplantation were studied. All patients were investigated in daily profile of tacrolimus blood levels. RESULTS: There was a strong correlation between area under the curve (AUC) and the trough concentration in both preprandial and postprandial oral administration, with correlation coefficients of 0.838 and 0.860, respectively. Significant increases in the peak concentration (Cmax) and AUC were observed in the preprandial oral administration subjects. Absorption of CAPD patients was obviously insufficient. CONCLUSIONS: Since tacrolimus absorption was better under fasted condition than under the presence of food, it is thought that the highest medicine effect with the least dosage is provided by preprandial oral administration. Furthermore, preprandial oral administration is recommended from a respect of economic burden reduction and side effect reduction.

Vascular access device for treatment of cancer patients.

Vascular access devices that are completely implanted have been used for treatment of cancer patients. Vascular access devices are useful for transarterial infusion of anticancer drugs, intravenous hyperalimentation, and drainage of bile juice in obstructive jaundice. These systems have several advantages in the care of patients: they are sealed, they have no external tubes, and they may be useful for blood or biliary sample and intravenous hyperalimentation or chemotherapy. There are only minimal discomforts related to the implantation procedure and no need for routine external catheter care. Most importantly, the quality of the patient's life is dramatically improved without external tubes. For these reasons, we believe that vascular access devices should be indicated for patients with malignant tumors as much as placement of these devices is technically feasible.

Prolongation of cardiac xenograft function after reduction of natural antibodies using double filtration plasmapheresis.

A double filtration plasmapheresis (DFPP) technique was applied to selectively remove the natural antibodies in discordant xenotransplantation. A swine heart was heterotopically transplanted in a canine neck after two DFPP procedures during which 200 to 500 ml of plasma were replaced with the same amount of Hartman's solution containing 7% human albumin. Mean removal rate of IgC and IgM by DFPP ranged from 74.2 +/- 0.5 to 95.9 +/- 2.8%. The anti-swine lymphocytotoxic reaction of canine serum was decreased after DFPP but still remained in low titers. Mean graft survival time in the group treated by DFPP in which 1000 ml of plasma was totally replaced was prolonged to 107 +/- 47 min, while it was 9 +/- 5 min in the group without any treatment (p less than 0.01). Deposits of canine IgM and C3 on the vascular endothelium of the graft were observed on immunofluorescence, while there were no deposits of IgG. In conclusion, DFPP effectively removed the natural antibodies, resulting in prolongation of xenograft survival time. Immunofluorescence study suggested that natural IgM antibodies played an important role in this xeno-hyperacute rejection.