Masaaki Kimikawa

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.

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.

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.

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.

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.

New CAPD catheter to prevent leakage of the dialysate.

CAPDカテーテル挿入術後の合併症であるカテーテル周囲からの灌流液漏れを根絶する目的で, 山本のカテーテル挿入法 (第1カフを腹膜で包み込む) を行った. さらに本術式をより簡便にするためにCAPDカテーテル (トラベノール社製) の第1カフの長さを短縮し, カテーテルの長軸方向に対して斜めに装着した新しいカテーテルを作製して, これを使用した. 昭和63年11月から平成元年5月の7か月間に10名の患者に対して12回のカテーテル植え込み術を行い経過を観察した.10名の患者 (男:女=7:3, 平均年齢: 49.2歳) に計12回の挿入術を施行したが, 第1カフの腹膜による包み込みは容易に行え, 挿入後の貯液量もスムーズに増加しえた. 術後の観察期間は平均76.6日 (7-180日) であるがカテーテル周囲からの灌流液漏れは1例もなく, 全例が術翌日から安静解除可能であった. しかし10例中2例はカテ先の位置異常により注排液が不良となり再挿入術を要した.山本の提唱したCAPDカテーテル植え込み法を追試し, これに著者らの作成したカテーテルを使用することにより, 手術操作をより簡略化できた.