Shuji Nobori

Marked prolongation of porcine renal xenograft survival in baboons through the use of α1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue

The use of animal organs could potentially alleviate the critical worldwide shortage of donor organs for clinical transplantation. Because of the strong immune response to xenografts, success will probably depend upon new strategies of immune suppression and induction of tolerance. Here we report our initial results using α-1,3-galactosyltransferase knockout (GalT-KO) donors and a tolerance induction approach. We have achieved life-supporting pig-to-baboon renal xenograft survivals of up to 83 d with normal creatinine levels.

Short- and long-term donor outcomes after kidney donation: analysis of 601 cases over a 35-year period at Japanese single center.

Background. The lack of deceased donors in Japan means that living-donor kidney transplantation is necessary in as many as 80% of cases. However, there are few data on perioperative complications and long-term outcome for live kidney donors. Methods. To determine associated perioperative morbidity and long-term mortality among live kidney donors, we reviewed 601 donor nephrectomies performed at our institution between 1970 and 2006 and attempted to contact all of the donors (or their families) to ascertain their present physical status. The survival rate and causes of death were compared with an age- and gender-matched cohort from the general population. Results. Although three donors (0.5%) experienced major perioperative complications, that is, femoral nerve compression, pulmonary thrombosis, and acute renal failure, all of the donors recovered and left hospital without complications. Among 481 donors (80%) for whom details were available at the time of inspection, 426 (88.5%) were still surviving. Donor survival rates at 5, 10, 20, and 30 years were 98.3%, 94.7%, 86.4%, and 66.2%, respectively. The mean interval between kidney donation and death was 183±102 (7–375) months, and the mean age at death was 70±11 years. The survival rate of kidney donors was better than the age- and gender-matched cohort from the general population, and the patterns and causes of death were similar. Conclusions. Our data suggest that continuation of living-donor kidney transplantation programs is justified in short- and long-term donor safety.

Thymic rejuvenation and the induction of tolerance by adult thymic grafts

The thymus, the site of origin of T cell immunity, shapes the repertoire of T cell reactivity through positive selection of developing T cells and prevents autoimmunity through negative selection of autoreactive T cells. Previous studies have demonstrated an important role for the thymus not only in central deletional tolerance, but also in the induction of peripheral tolerance by vascularized renal allografts in juvenile miniature swine recipients. The same protocol did not induce tolerance in thymectomized recipients nor in recipients beyond the age of thymic involution. We subsequently reported that vascularized thymic lobe grafts from juvenile donors were capable of inducing tolerance in thymectomized juvenile hosts. However, the important question remained whether aged, involuted thymus could also induce tolerance if transplanted into thymectomized hosts, which, if true, would imply that thymic involution is not an intrinsic property of thymic tissue but is rather determined by host factors extrinsic to the thymus. We report here that aged, involuted thymus transplanted as a vascularized graft into juvenile recipients leads to rejuvenation of both thymic structure and function, suggesting that factors extrinsic to the thymus are capable of restoring juvenile thymic function to aged recipients. We show furthermore that rejuvenated aged thymus has the ability to induce transplant tolerance across class I MHC barriers. These findings indicate that it may be possible to manipulate thymic function in adults to induce transplantation tolerance after the age of thymic involution.

Management and outcome of living kidney grafts with multiple arteries.

PurposeKidney allografts with multiple renal arteries (MRAs) have been used with increasing frequency since the advent of laparoscopic live donor nephrectomy. To determine if MRA grafts affect the short- and long-term outcomes of grafts and patients, we analyzed 340 grafts procured by open nephrectomy.MethodsWe divided the graft recipients into five groups according to the methods used for vascular reconstruction. We compared patient and graft survival, serum creatinine levels, total (rewarm) ischemic times (TIT), incidence of acute tubular necrosis (ATN), need for antihypertensive drugs, incidence of acute rejection episodes, and vascular and urologic complications, between the MRA group and a control group of patients with single-artery renal grafts.ResultsIn patients who underwent multiple anastomoses in situ, prolonged TIT resulted in an increased incidence of ATN, but there was no significant difference between the MRA groups and the control group (P = 0.45). The incidence of vascular complications was higher in the MRA groups (P < 0.01), but there were no significant differences in the other variables among the groups.ConclusionMultiple renal artery grafts procured by open nephrectomy can be transplanted as successfully as those with single arteries, by using meticulous suturing techniques.

Expression of Cyclooxygenase-1 and -2 in Human Breast Cancer

AbstractPurpose. We investigated the expression of cyclooxygenase (Cox-1 and Cox-2) in mammary tissues from patients with breast cancer. Methods. We used reverse transcriptase–polymerase chain reaction (RT-PCR) and immunohistochemistry. Results. The cancer cells showed very weak expression of Cox-1, but strong expression of immunoreactive Cox-2. In contrast, immunoreactive Cox-2 was very weak in all of the benign mammary tumors examined, including fibroadenoma (FA) and mastopathy (MP). Immunoreactive Cox-1 was also very weak in these benign tumors. The extent and intensity of immunoreactive Cox-2 polypeptides was significantly greater in the cancer cells than in the FA cells or MP cells. RT-PCR analysis showed enhanced expression of Cox-2, but not Cox-1 in breast cancer tissue, and faint expression of Cox-2 in benign tissue. Conclusions. These results demonstrated that human breast cancer cells generated Cox-2, indicating that Cox-2 might play an important role in the proliferation of breast cancer cells.

Long-term acceptance of fully allogeneic cardiac grafts by cotransplantation of vascularized thymus in miniature swine.

Background. We have previously reported the ability of both thymokidney and vascularized thymic lobe (VTL) allografts to induce transplantation tolerance to renal allografts across a full major histocompatibility complex (MHC) mismatch in thymectomized miniature swine. However, whether vascularized thymus is capable of inducing tolerance to less tolerogeneic organs when it is transplanted simultaneously is not yet known. The present study investigates cardiac allograft survival and the mechanism of long-term acceptance in recipient swine following cotransplantation of VTL and cardiac grafts from fully MHC-mismatched donors. Methods. Animals received a heart graft, a heart graft and a VTL, or a heart graft and a donor thymocyte infusion. Immunosuppressive regimens consisted of 12 or 28 days of tacrolimus. Results. All animals that received a VTL maintained their grafts significantly longer than their counterparts that received only a heart graft, and those receiving 28 days of tacrolimus maintained their heart grafts long-term. Recipients of a donor thymocyte infusion demonstrated slightly prolonged cardiac graft survival but all rejected their grafts, highlighting the importance of thymic stroma. Cytotoxic T-lymphocyte responses against third-party antigens by cells from tolerant animals showed restriction by both self and donor MHC, whereas responses of controls were restricted to self MHC only. The presence of donor dendritic cells in the VTL grafts and results of co-culture assays suggest that both central and regulatory mechanisms were involved in achieving long-term acceptance. Conclusion. This is the first demonstration of the long-term acceptance of fully MHC-mismatched cardiac allografts in large animals.

Role of Persistence of Antigen and Indirect Recognition in the Maintenance of Tolerance to Renal Allografts

Background. We have previously shown that a 12-day treatment with cyclosporine A (CyA) facilitates induction of tolerance to class-I disparate kidneys, as demonstrated by acceptance of second, donor-matched kidneys without immunosuppression. In the present study, we have examined 1) the duration of tolerance in the absence of donor antigen and 2) the pathway of antigen recognition determining maintenance or loss of tolerance. Methods. Seventeen miniature swine received class-I mismatched kidneys with 12 days of CyA, and received second donor-matched kidneys without immunosuppression at 0, 1, 3, or 4 months after nephrectomy of the primary graft. Five were sensitized 6 weeks after nephrectomy of the primary graft, three with donor-matched skin grafts, and two with donor class-I peptides to eliminate direct pathway involvement. In addition, two long-term tolerant animals received class-Ic peptides. Results. Rejection of second grafts required at least a 3 month absence of donor antigen. Although donor-matched skin grafts in animals tolerant to kidneys induced antidonor cytotoxic T lymphocyte responses, second renal transplants revealed no evidence of sensitization. In contrast, immunization of recipients with donor class-I peptides after nephrectomy of the primary graft led to loss of tolerance at both T-cell and B-cell levels, as evidenced by rejection of the second graft in 5 days and development of antidonor immunoglobulin G. Peptide immunization of long-term tolerant in recipients bearing long-term renal grafts did not break tolerance. Conclusions. These data indicate that the renal allograft is required for the indefinite maintenance of tolerance, that indirect antigen presentation is capable of breaking tolerance, and that in tolerant animals, direct antigen presentation may suppress rejection, allowing tolerance to persist.

The Consequences for Live Kidney Donors With Preexisting Glucose Intolerance Without Diabetic Complication: Analysis at a Single Japanese Center

Background. Because of lack of deceased donors in Japan, there has been a need to expand the eligibility criteria for live kidney donation. To assess the indications for live kidney donation in glucose intolerance (GI), we analyzed perioperative complications associated with donor nephrectomies performed at our institution and followed up the long-term consequences. Methods. The 444 live kidney donors were divided into two groups based on the results of the 75-g oral glucose tolerance test: a GI group (n=71) who showed a diabetic (n=27) or impaired glucose tolerance (n=44) pattern, and a non-GI group (n=373) who showed a normal oral glucose tolerance test pattern. Perioperative complications, long-term survival rate, and frequencies of hypertension, diabetes, hyperlipidemia, and renal dysfunction in long term were compared in each group. Results. The incidence of perioperative complications was not higher in the GI group than in the non-GI group (4.3% vs. 5.4%, respectively; NS). Survival rates in the GI group at 5, 10, and 20 years were 98.3%, 95.1%, and 89.2%, respectively, whereas those in the non-GI group were 98.0%, 96.1%, and 91.5%, thus showing equivalent mortality. None of the patients in the diabetes mellitus group had developed severe diabetic complications or end-stage renal disease at a mean follow-up point of 88±71 (range, 14–225) months. Conclusions. Our results suggest that individuals who have GI without diabetic complication may be able to donate their kidney safely with little surgical complication and little major morbidity if strict evaluation is performed before transplant.

The Efficacy and Safety of High-Dose Mizoribine in ABO-Incompatible Kidney Transplantation Using Anti-CD20 and Anti-CD25 Antibody Without Splenectomy Treatment

BACKGROUND Mizoribine (MZR) has been developed as an immunosuppressive agent in Japan, but it shows less potent immunosuppressive effects at doses up to 3 mg/kg/d. In this study, we investigated whether high-dose MZR (6 mg/kg/d) was effective for ABO-incompatible (ABO-i) living donor kidney transplantation (LKT) using treatment with anti-CD25 and anti-CD20 monoclonal antibodies without splenectomy. METHODS Since 2007, we encountered 24 cases of ABO-i LKT using anti-CD20 and anti-CD25 monoclonal antibody without splenectomy. The pretransplant immunosuppressive regimen consisted of two doses of anti-CD20 antibody, mycophenolate mofetil (MMF), prednisolone, a calcineurin inhibitor (cyclosporine [7 mg/kg] or tacrolimus [0.2 mg/kg] and two doses of anti-CD25 antibody. Antibody removal by plasmapheresis was performed before LKT up to several times according to the antibody titer. The posttransplant regimen consisted of high-dose mizoribine (6 mg/kg/d) instead of MMF (MZR group, n = 12). RESULTS The 1-year graft survival rates for the MZR and MMF groups were both 100%. The rejection rate in the MZR group (eight %) was not significantly higher than that in the MMF group (seventeen %) Serum creatinine level was not significantly different between the two groups. In the MZR group 6 (50%) patients developed CMV antigenemia-positivity versus 11 (92%) in the MMF group (P < .05). The number of patients who developed CMV disease was 0 in the MZR group and 1 (8%) in the MMF group. The number of patients treated with ganciclovir was 0% and 8%, respectively (not significant). CONCLUSIONS We obtain good clinical results with high-dose MZR in ABO-i LKT using anti-CD20 and anti-CD25 antibody treatment without splenectomy.

Reversal of age-related thymic involution by an LHRH agonist in miniature swine.

UNLABELLED BACKGROUND AND AIMS OF STUDY: We have previously demonstrated a requirement for the presence of a juvenile thymus for the induction of transplantation tolerance to renal allografts by a short-course of calcineurin inhibition in miniature swine. We have also shown that aged, involuted thymi can be rejuvenated when transplanted as vascularized thymic lobes into juvenile swine recipients. The present studies were aimed at elucidating the extrinsic factors facilitating this restoration of function in the aged thymus. In particular, we tested the impact of sex steroid blockade by Luteinizing Hormone-Releasing Hormone (LHRH). MATERIALS AND METHODS 30 naive animals (25 males and 5 females) were used for measurement of serum testosterone levels. 3 mature male pigs (aged at 22, 22 and 29 months old) were used to test the effects of Lupron (LHRH analog) injection at 45 mg (per 70-80 kg body weight) as a 3-month depot on testosterone levels and thymic rejuvenation. Thymic rejuvenation was assessed by histology, flow cytometric analysis, morphometric analysis and TREC assays. RESULTS Hormonal alterations were induced by Lupron and resulted in macroscopic and histologic regeneration of the thymus of aged animals within 2 months, as evidenced by restoration of juvenile thymus architecture and increased cellularity. Two animals that were evaluated for TREC both showed increased levels in the periphery following Lupron treatment. CONCLUSION Treatment of aged animals with Lupron leads to thymic rejuventaion in adult miniature swine. This result could expand the applicability of thymus-dependent tolerance-inducing regimens to adult recipients.