Clinical Small Bowel Transplantation Research Articles

Impact of Antimouse CD52 Monoclonal Antibody on Graft’s γδ Intraepithelial Lymphocytes After Orthotopic Small Bowel Transplantation in Mice

Alemtuzumab, a humanized CD52 monoclonal antibody (mAb), has been used for immune induction in clinical small bowel transplantation (SBT). However, the local impact of CD52 mAb on the transplanted intestine is not known. In this study, we investigated the effects of CD52 mAb on the intestinal intraepithelial lymphocytes (iIELs) in grafts using a murine orthotopic SBT model. The antimouse CD52 mAb was used as a surrogate antibody. Graft rejection was assessed by histopathologic analysis, iIELs were isolated for flow cytometric analysis, the mucosal keratinocyte growth factor (KGF) expression was evaluated by real-time polymerase chain reaction, and epithelial cell (EC) proliferation (percent bromodeoxyuridine incorporation) was measured by immunohistochemistry. No mice showed evidence of rejection. Seven days after SBT, when the CD52 mAb was used, the numbers of iIELs and both T-cell receptor (TCR) γδ and TCRαβ subpopulations were decreased, and the number of TCRγδ iIELs of donor origin was prominently reduced in the allografts. Furthermore, TCRγδ iIEL-derived KGF messenger RNA expression and EC proliferation decreased significantly after CD52 mAb application. These data demonstrate that the antimouse CD52 mAb could deplete iIELs in the transplanted intestine, especially the TCRγδ subset of donor origin. The reduction of TCRγδ iIELs accompanied the decrease in mucosal KGF expression and EC proliferation, which may slow down the epithelial turnover and retard the repair of the damaged epithelium.

Combination Therapy of Tacrolimus and Infliximab Reduces Inflammatory Response and Dysmotility in Experimental Small Bowel Transplantation in Rats

Intestinal transplantation initiates a functionally relevant inflammatory response by activation of resident macrophages within the muscularis associated with dysmotility. Infliximab is used successfully as a potent anti-inflammatory agent for the treatment of chronic inflammatory bowel diseases and as rescue therapy in acute steroid-resistant rejection in selected settings in clinical small bowel transplantation. We hypothesize that additional perioperative treatment with infliximab diminishes initiation of the inflammatory cascade and improves motility in small bowel grafts using a standard tacrolimus immunosuppressive protocol. Orthotopic intestinal transplantation was performed in rats. In two treatment groups (24/168 hr), infliximab was administered intravenously directly after reperfusion and tacrolimus was injected intramuscularly after transplantation and once a day. Two other treatment groups (24/168 hr) received standard immunosuppressive therapy with tacrolimus. Isogenic and allogenic transplanted vehicle-treated animals (24/168 hr) and native gut served as control. Infliximab-treated grafts exhibited significantly less leukocyte infiltration at 24/168 hr after transplantation and at 168 hr significantly less apoptosis in the tunica muscularis compared with tacrolimus monotherapy. Additional infliximab treatment resulted in increased smooth muscle contractility (30%) after 24 hr compared with tacrolimus control. Dysmotility of transplanted small bowel results from reperfusion injury and acute rejection. Additional perioperative treatment with infliximab reduces early unspecific inflammatory responses and complements immunosuppressive therapy with tacrolimus.

Richard Carlton Lillehei Transplant and Shock Surgical Pioneer

ABSTRACTRichard Carlton Lillehei (1927–1981) was a trainee of the Minnesota School of Surgery under Owen H. Wangensteen in the early 1950s, when great advances were being accomplished in cardiac surgery. His mentors besides Wangensteen included his brother C. Walton Lillehei, Richard L. Varco, and several others in the Department of Surgery. His interest in surgical research was evident since the early part of his training. The understanding of the nature and management of shock occupied his attention and persisted all his life. Equally, his studies on organ preservation and intestinal and pancreatic transplantation were classic in the evolution of surgery. In fact, Rich Lillehei, together with William Kelly, performed the world's first clinical pancreas transplant in 1966 and with his own team, the world's first clinical small bowel transplantation in 1967. Following that, these two areas of transplantation progressively advanced to be applied to thousands of patients worldwide. Lillehei was also a committed teacher and many of his students are disseminated throughout the globe. Unfortunately, his enormous contributions were cut short with his premature death at age 53. However, his surgical legacy remains alive!

Recovery of allografted small intestine function.

To investigate recovery of the allografted small intestine function after clinical small bowel transplantation (SBT). The structure of the graft was evaluated by endoscopic biopsy and histopathologic examination. Graft functions were assessed by D-xylose absorption, barium studies, nitrogen balance calculation, and blood and stool cultures. Nutritional status of the recipients was judged by measurement of body weight and serum protein concentrations. The recipient discontinued total parenteral nutrition (TPN) and resumed oral nutrition 100 d after SBT. On oral diet, the patient maintained a normal nutritional status, gained weight by 3 kg, and had a normal serum albumin concentration (40.2 g/L ± 0.2 g/L). Satisfactory D-xylose absorption was achieved 8 wk after the operation. Nitrogen balance of the gut was maintained well and increased gradually. Serial mucosal biopsy showed normal structures 2 wk after grafting, without evidence of rejection and graft versus host diseases (GVHD). Barium studies conducted on the 10(th) day and 38(th) day by barium studies revealed that the grafted small bowel motility showed normal patterns of peristalsis and transit. No bacterial translocations were noted. Function of the grafted small intestine recovered satisfactorily 100 d after transplantation, indicating good clinical outcome of SBT.

Ischemic preconditioning of small bowel mitigates the late phase of reperfusion injury: heme oxygenase mediates cytoprotection

Background Ischemia and reperfusion (IR) injury of the intestine is a major cause of morbidity and mortality following small bowel transplantation. The current study evaluates the effect of ischemic preconditioning (IPC) on the intestinal microcirculation in the late phase of IR injury of the intestine. Methods Sixty rats were randomly allocated to 5 study groups (n = 12 per group): (1) sham, (2) IR (3) IPC, (4) pyrrolidine dithiocarbamate (PDTC) (HO-1 inducer), and (5) zinc protoporhyrin (ZnPP) (HO-1 inhibitor). Mucosal perfusion and leukocyte–endothelial interactions were measured with the aid of an intravital microscope. At the end of the experiments, blood samples for lactate dehydrogenase (LDH) levels and biopsies of ileum for histologic evaluation were obtained. Results IPC significantly improved the mucosal perfusion and decreased the leukocyte–endothelial interactions. Histologic examination showed that ileal mucosa was significantly less injured in the IPC and PDTC groups as compared with the IR group. Conclusions IPC protects the intestine from late reperfusion injury. HO-1 is involved in this protection. These findings may be of significant importance in clinical small bowel transplantation.

Short-Term Anti-CD4 Plus Anti-TNF-α Receptor Treatment in Allogeneic Small Bowel Transplantation Results in Long-Term Survival

Despite improved immunosuppression, intestinal transplantation is still complicated by severe rejection episodes. To further improve immunosuppressive concepts, we evaluated an anti-CD4 antibody and an anti-tumor necrosis factor (TNF)-alpha monoclonal antibody for their immunosuppressive efficacy in the standard rat model of intestinal transplantation. Intestinal transplantation was performed in the DA to Lewis combination, and recipients were treated perioperatively with either the anti-CD4 antibody RIB5/2 (day -1, 0, postoperative days 1, 2, 4, 7, 10, 14, 17, and 21), the anti-TNF antibody etanercept (60 min before reperfusion, postoperative days 3, 6, and 9) or a combination of both. Survival, histology and expression of immunologic mediator genes on days 3 and 4 after transplantation were investigated. Treatment with anti-CD4 antibody alone (19.71+/-5.94) and the antibody combination (171.58+/-122.76) prolonged survival. The chemokine MIP-1alpha was significantly decreased in both anti-CD4 antibody treatment groups, possibly indicating an additional effect of the TNF-alpha blockade on the immune modulation by RIB5/2. Our study demonstrated long-term graft survival in short-term treatment with a combination of an anti-CD4 antibody and a TNF-alpha antibody in more than 50% of the recipients of intestinal grafts. Such a combined approach could also be useful in clinical small bowel transplantation.

Intragraft Gene Expression Profile During Acute Cellular Rejection in Clinical Small Bowel Transplantation: A Case Report

Background Acute cellular rejection (ACR) is a common complication of small bowel transplantation (SBTx) and the major cause of graft loss. However, little is known regarding the genetic graft response to ACR in clinical transplants. In this study, we have determined a genetic expression profile of intestinal graft response to ACR after living related (LR) SBTx. Results By identifying the expression profiles of reported markers of rejection we were able to identify 57 genes that had significantly increased (more than twofold) expression in response to ACR. Known markers of rejection identified: MMP-9, MMP-2, VIP, IFNγ, IL-2R, MADCAM-1, HSP-60, and HSP-70 all had greater than twofold increased expression after ACR diagnosed (week 3 to week 6). The newly identified genes were: IFI27, EPST11, APAF1, LAP3, STK6, and MDK. Conclusion Newly identified up-regulated genes in response to ACR in small bowel graft are involved in the immune response, cell adhesion, neurogenesis, cell division and proliferation, DNA replication/repair, protein ubiqutin/proteolysis, and apoptosis. TNFα up-regulated early at week 2 biopsy may be an early genetic marker of ACR in SBTx.

Early Outcome of Different Steroid-Free Regimens in Small Bowel Transplantation: A Large-Animal Study

The intestine is a highly immunogenic organ that requires heavy immunosuppression (IS); therefore corticosteroid withdrawal after clinical small bowel transplantation (SBT) has not been standardized. In this study, we compared different immunosuppressive regimens (none with steroid or induction treatment) in a SBT pig model. Large White unrelated piglets were transplanted and divided into four groups as follow: group 1 ( n = 3): no IS; group 2 ( n = 10): IS with tacrolimus only; group 3 ( n = 10): IS with tacrolimus and mycophenolate mofetil; group 4 ( n = 5): IS with tacrolimus and rapamycin. Follow-up time was 30 days. All IS drugs were given orally; tacrolimus whole blood levels ranged between 5 and 15 ng/mL in all groups except for group 2 whose tacrolimus whole blood levels ranged between 15 and 25 ng/mL. Group 1 pigs died of graft acute rejection (ACR) after a median of 12 days. Overall survival in groups 2, 3, and 4 at day 30 was 40%, 80%, and 60%, respectively. Biochemical parameters, including glycemia and cholesterol, were into the normal range with no significant differences between groups. At the end of the study, one animal in group 2 and another one in group 4 showed histological signs of moderate to severe ACR. The incidence of infection was higher in group 2 (2.1 episodes/pig) compared to group 3 (1.25) and group 4 (1.6). This large-animal study demonstrates that tacrolimus-based IS without corticosteroids allows, in the early postoperative period (30 days) after SBT, good survival rates without an increased risk in the incidence of rejection.

Incidence of Graft Rejection in Small Bowel Transplanted Pigs After Immunosuppression Withdrawal

As intestinal grafts require heavy immunosuppression, there are no reports of immunosuppression withdrawal after clinical small bowel transplantation. In this large-animal study, we investigated the occurrence of graft rejection in intestinal-transplanted pigs after withdrawal. Large-White unrelated piglets were transplanted and divided in three groups: group 1 ( n = 5), intestinal transplantation (ITx) with no immunosuppression; group 2 ( n = 7), Itx and 60 days of treatment with tacrolimus and mycophenolate mofetil; group 3 ( n = 5), Itx and donor bone marrow infusion (DBMi) and 60 days of treatment with tacrolimus and mycophenolate mofetil. Follow-up time after withdrawal was 120 days. Group 1 pigs died of graft acute cellular rejection (ACR) after a median of 11 days. In group 2, two pigs died of ACR-related infection and another two of ACR within 90 days. The remaining three animals (43%) were sacrificed at day 180, and their grafts showed no signs of ACR. In group 3, two pigs died of ACR-related infection and one of graft versus host disease within 80 days; at day 180 the two surviving animals showed signs of chronic rejection in the allograft. This study demonstrates that total withdrawal after ITx is followed by sudden and lethal ACR (or ACR-related infection) in more than 50% of the recipients. When a tolerance-inducing strategy as DBMi is applied, lethal graft versus host disease may also occur. In group 3, the intestinal allograft, to which the recipients were partially tolerant, developed chronic rejection that was probably associated with a decline with time of donor-leukocytes chimerism, as recently demonstrated in rats.

Efeitos hemodinâmicos e metabólicos iniciais da perfusão hipotérmica intestinal in situ.: avaliação de um novo modelo canino de autotransplante intestinal

O transplante intestinal tem sido utilizado como uma alternativa promissora no tratamento da síndrome do intestino curto em pacientes adultos e pediátricos. Vários modelos experimentais foram desenvolvidos com a finalidade de testar diferentes soluções de preservação. No entanto são modelos complexos que levam, muitas vezes, um tempo prolongado para a sua realização. OBJETIVO: Neste estudo, desenvolvemos um modelo alternativo de autotransplante intestinal in situ analisando os efeitos hemodinâmicos e metabólicos iniciais da perfusão hipotérmica com solução de Ringer lactato. MÉTODOS: Foram utilizados seis cães machos sem raça definida (22,8±1,4 Kg); as variáveis hemodinâmicas sistêmicas foram obtidas por meio de cateter arterial e pelo Swan-Ganz. A perfusão do trato gastrintestinal foi avaliada pela medida do fluxo sangüíneo da veia mesentérica superior (FSVMS, fluxômetro ultra-sônico), e através da medida do pCO2 intestinal (pCO2-int e pCO2-gap, tonometria a gás). Inicialmente realizamos a secção do jejuno proximal e íleo distal e isolamento dos vasos mesentéricos com fitas cardíacas, todo o tecido nervoso e linfático em torno do eixo vascular intestinal foi seccionado. O território mesentérico foi perfundido através da artéria mesentérica superior por 30 minutos com 1000 ml de Ringer lactato a 4ºC, e o efluente drenado através de uma pequena incisão na veia mesentérica superior. Os animais foram observados por 120 min após o inicio do período de reperfusão. Amostras de sangue foram obtidas da aorta abdominal, para análise gasométrica. RESULTADOS: A perfusão intestinal hipotérmica induziu uma redução do FSMVS apenas nos primeiros 30 min de reperfusão (587±70,9 para 398±102,8 ml/min) e um aumento do pCO2-gap (2±2,7 para 29,8±6 mmHg). Não foram observadas alterações significativas em relação a parâmetros hemodinâmicos e metabólicos sistêmicos (PAM, DC, pH, excesso de bases, hemoglobina) assim como na temperatura central. CONCLUSÃO: O modelo de autotransplante intestinal é extremamente útil e de fácil execução, para a avaliação inicial de soluções de preservação e/ou drogas antioxidantes, comumente utilizadas no transplante de intestino.

Small intestine transplantation

Significant progress has been made in clinical small bowel transplantation over the last decade mainly due advances in biotechnology and new immunosuppressive regiments. This transplantation has now been indicated to treat special cases of intestinal failure. This review highlights recent developments in the area of small bowel transplantation. Over 600 reports on clinical and experimental small bowel transplantation were reviewed. Aspects concerning research development, different immunosuppressive strategies, patient and graft monitoring, and improvements in surgical techniques are discussed. About 700 small bowel transplantation were performed in 55 transplant centers, 44% intestine-liver, 41% isolated intestinal graft and 15% multivisceral transplantation. Rejection and infection are the main limitation of this procedure. Actual 5 years post transplantation graft survival of the total international experience is 46% for isolated intestinal graft, 43% for combined intestine-liver and nearly 30% for multivisceral transplantation. Higher graft and patient survival are seen at the more experienced centers. In a series of 165 intestinal transplantation at University of Pittsburgh, PA, USA, actuarial patient survival was reported to be over 75% at one year, 54% at 5 years and 42% at 10 years. Over 90% patients from Pittsburgh program resume an unrestricted oral diet. Small bowel transplantation has advanced from an experimental strategy to a feasible alternative for patients with permanent intestinal failure. Further refinements in graft acceptance, immunosuppressive regiments, infection management and prophylaxis, surgical techniques as well as appropriated patient referral and selection are crucial to improve outcomes.

Small bowel transplantation - current status and initial results

During the last years, clinical small bowel transplantation has significantly improved. This is especially true for isolated small bowel transplantation with success rates of 80 % 1-year patient survival. Currently, approximately 100 small bowel transplantations are performed per year worldwide. In Germany, small bowel transplantation is still rare, because of the high risk for the development of acute rejection and rejection-associated complications. This includes peritonitis and sepsis as well as over-immunosuppression-associated infections. Due to improvements in immunosuppression, the incidence of acute rejection decreased from about 85 % below 25 %. Half of the patients will receive a combined liver-small bowel graft due to TPN (total parenteral nutrition)-associated liver cirrhosis. Although the combined procedure has immunological advantages, complication rates are high and patient survival is significantly lower (ca. 50 % at 1 year). Next to bacterial, fungal, and atypic infections, which are frequently associated with rejection and other complications, CMV and EBV infections are of significant interest. This is of special importance for EBV infections, since all PTLD (lymphoproliferative disease) after small bowel transplantation are EBV-associated so far. Viral infections should be monitored and preemptive therapy using ganciclovir or foscavir will be initiated. Of the 800 patients transplanted so far, 50 % are still alive up to 15 years. Of these, more than 80 % are off parenteral nutrition, in good healths, with good quality of live.

Modified surgical technique in clinical small bowel transplantation: donor and recipient management

Modified surgical technique in clinical small bowel transplantation: donor and recipient management

Clinical small bowel transplantation: focus on mucosal barrier function

RECENTLY SMALL BOWEL transplantation has evolved to an established treatment for patients with short gut syndrome and intestinal failure. The introduction of new immunosuppressants offer the opportunity to minimize acute rejection episodes and associated problems. Nevertheless, good initial graft function with maintainance of the mucosal barrier function are of great importance. Preservation/reperfusion injury impairs mucosal barrier function and furthermore initiates the release of mediators. The release of specific and nonspecific inflammatory mediators may promote serious postoperative complications including the development of infection and acute rejection. Therefore, new strategies are required to decrease reperfusion injury and restore mucosal barrier function. Early postoperative enteral nutrition has been shown to restore mucosal barrier function and to decrease the incidence of infection including pneumonia, peritonitis, and sepsis. Enteral preparations containing glutamine, arginine, and omega-3 fatty acids, so called immunonutritions, are known to decrease the release of cytokines, reactive oxygen intermediates, and other mediators in critically ill patients. Lactobacilli, naturally found in the small intestine, have been shown to prevent bacterial overgrowth, to restore gut barrier function, and thereby, to reduce the occurrence of infection. To decrease infectious complications and possibly rejection, donors and recipients of small bowel grafts were treated with lactobacilli and immunonutrition.

Tetracycline prophylaxis abrogates mortality following induction of inferior vena cava sepsis

DISRUPTION OF THE intestinal barrier following small bowel transplantation (SBT) has been associated with ischemia-reperfusion, cold storage, rejection, and immunosuppressive regimens. The altered intestinal barrier, consequent to SBT, has been shown to be susceptible to the occurrence of increased bacterial translocation (BT). In addition, increasing evidence has implicated BT as the main source of postoperative infection. The major problem with severe infection has been related to the overwhelming production of proinflammatory mediators. According to recent studies, antibiotics of the tetracycline (TET) family have been credited to have anti-inflammatory properties, not related to their antimicrobial efficacy but to their ability to down-regulate protein kinase C activity, LPS-induced serum TNF, and also by protecting mice against LPS-induced lethality. Other researchers have suggested that the direct inhibition of nitrate release is the main mechanism of the anti-inflammatory activity of doxycicline in septic shock. Considering that in clinical small bowel transplantation, the graft venous drainage can be directed either to the portal (P) or to inferior vena cava (IVC), we proposed to investigate the possible beneficial effect of tetracycline prophylaxis, using an IVC bacterial sepsis model. In our previous studies, an IVC sepsis model was used to simulate the IVC via of BT with known bacterial concentrations.

Substantial changes in the intestine-derived lymph during bacterial translocation

NFECTION CONTINUES to be an important concern in clinical small bowel transplantation (SBT), and increasing evidence has credited bacterial translocation (BT) as being a possible source for the pathogenesis of postsurgical infection. 1– 4 In addition, the so-called gut hypothesis of the pathogenesis of sepsis progressing to multiple organ failure has been attributed to enhanced BT in septic states by increasing vascular permeability. 5,6 In previous works, we have shown two distinctive routes of BT, lymphatic and hematological. 7 However, the lymphatic via only showed positive BT up to the mesenteric lymph node (MLN) through the intestinal afferent ducts, and, surprisingly, negative BT by efferent mesenteric lymph duct (MLD). 7 Thus, we raised the hypothesis that the lymphatic via of BT was possibly linked to host immune activation and the hematological via would be the true route for intestinal bacteria to reach the systemic circulation. In this study, we investigated the role of the lymphatic via of BT on host immune response by examining changes in efferent MLD contents using a previously established BT model in rats. 2

Monitoring of immunosuppression after clinical small bowel transplantation

ACUTE REJECTION (AR) remains the most prominent risk factor following small bowel transplantation. Despite improved patient and organ survival rates from approximately 50% to 97% and 71%, respectively, by switching immunosuppression from cyclosporine (CsA)based protocols to tacrolimus (Tac)-based regimens, the rejection rate remains as high as 95% with 60% of rejection episodes occurring in the initial 3 months. New potent immunosuppressants, such as rapamycin (Rapa) and monoclonal antibodies against IL-2 receptor (daclizumab [Dac]), are believed to have the potential to further decrease rejection rates and improve outcomes after small bowel transplantation (SBTx). Pinna et al compared different induction protocols, including cyclophosphamide, mycophenolate mofetil, OKT3, and Dac, with Dac resulting in slightly decreased rejection rates and fewer infectious complications. Abu-Elmagd et al presented preliminary data suggesting the efficacy of Dac as induction therapy together with a Tac-based immunosuppressive regimen to reduce acute rejection (AR) rates (by 43%). The incidence rate of viral infection for and posttransplant lymphoproliferative disease was 7% for each. The impact of additional to Tac-based protocols of Rapa, steroids, and Dac induction therapy is undergoing evaluation in terms of whether they decrease the AR rate while increasing the risk for development of immunosuppression-associated infections, including CMV and EBV. However, monitoring of immunosuppression may facilitate individualized rather than fixed dosing of induction therapy with Dac.

Use of inferior mesenteric vein for venous anastomosis in clinical small bowel transplantation

Use of inferior mesenteric vein for venous anastomosis in clinical small bowel transplantation

Effect of epidermal growth factor on intestinal adaptation after allogeneic small bowel transplantation in rats

We reported that epidermal growth factor (EGF) stimulated graft adaptation in a rat model of syngeneic small bowel transplantation. However, graft rejection is a severe problem with clinical small bowel transplantation, because small intestinal wall contains large amounts of lymphoid tissue. Studies were performed to investigate the effect of EGF on allogeneic graft adaptation after small bowel transplantation in rats treated with an immunosuppressant FK506. The transplanted animals received intraperitoneally EGF or saline (untreated) after surgery and were examined for analysis one week later. EGF-treated group markedly enhanced the water absorption and induction of sodium glucose cotransporter (SGLT1) as compared with EGF-untreated group. EGF-treated group also increased the mucosal crypt depth and its cell proliferating rate, although there was no significant difference in the mucosal villus height between the two groups. These results indicate that EGF accelerates intestinal allograft adaptation in part by the recovery of mucosal structure and function after small bowel transplantation in rats. EGF may have relevance to promote graft function in clinical small intestinal transplantation.

Low infectious complications in segmental living related small bowel transplantation in adults

Clinical small bowel transplantation (SBTx) has been associated with a high rate of infectious complications. Laparotomy, preservation injury, abnormal motility, lymphatic disruption, aberrant systemic venous drainage, rejection and antibiotic therapy could all be implicated in the etiology of these complications. In addition to the underlying disease, total parental nutrition could determine infections and liver impairment. Recently, standardized techniques for segmental living related SBTx (LR-SBTx) have been developed. This technique allows reduction of some of these factors, thus resulting in a reduced incidence of infections. We report the infectious complications observed in 3 patients with short bowel syndrome treated with LR-SBTx at our institution. A segment of 180-200 cm of ileum was transplanted with a neglectably short cold ischemia time (CIT). The donor bowel was decontaminated. Oral tacrolimus, prednisone and IV induction with ATG were used for immunosuppression. Blood, stool, urine, sputum and peritoneal fluids were collected and cultured as a routine surveillance. All recipients are alive with a current follow-up time up to 21 months. No bacterial infections were observed during the post-transplant period. One patient developed cytomegalovirus (CMV) enteritis 4 months after LR-SBTx and was treated successfully with IV ganciclovir. In our limited experience, LR-SBTx is associated with a low infection rate. This could be due to optimal graft decontamination, short CIT and to the reduced incidence of rejection and lower immunosuppression used in this immunologically well-matched combination.