Fetal pancreas transplantation in miniature swine. I. Developmental characteristics of fetal pig pancreases

The genetically defined miniature pig developed by Sachs et al. was selected as a large animal model to test the feasibility of fetal pancreas transplants for reversal of insulin-dependent diabetes. In order to test our approach, the first key was to characterize the development of the pig pancreas tissues throughout fetal life. Pancreas samples were obtained from 102 farm pig fetuses ranging in age from 35-110 days and from 39 minipig fetuses removed by Caesarean section from 5 timed-pregnant sows between 33 and 73 days after conception. The development of the endocrine and exocrine pancreases were examined by immunobiochemical assays of insulin, chymotrypsinogen, and trypsinogen. Light and electron microscopic examination of pancreases from the critical fetal ages (35-55 days) were used to confirm the above results. Insulin was already present at day 33 and increased rapidly till birth (day 114: 2.2 U/pancreas). Chymotrypsinogen activity was first detected at day 43 and trypsinogen activity at days 49-50. Enzyme content increased rapidly till days 65-70 and then more gradually until birth. Morphological development of exocrine cell granules conformed to the above results. The results clearly demonstrated in the pancreas of a larger mammal, the pig, that the endocrine elements mature prior to the exocrine system. Thus, as we found in rats, pig fetal pancreas also has this advantage as a donor tissue for transplantation. An appropriate fetal age for pig donors is estimated to be between 45 and 50 days.     

Bone marrow transplantation in miniature swine. I. Development of the model

Procedures for successful autologous and MHC-matched allogeneic bone marrow transplantation in partially inbred, MHC-defined miniature swine have been established. All marrow recipients were conditioned with single-dose total-body irradiation at the upper level of tolerance, and supported with antibiotics and irradiated blood products during aplasia. Surgical harvest of autologous and allogeneic marrow yielded sufficient numbers of cells to successfully reconstitute recipients. Radiation control animals that received no marrow failed to show recovery of marrow function. Pigs transplanted with autologous marrow at doses greater than 10(8) cells/kg routinely engrafted and recovered normal marrow function. The major clinical complications were acute and chronic infections and hemorrhage. T cell-depleted autologous marrow also engrafted, and there was no observed increase in clinical complications. In bone marrow transplantation across non-MHC allogeneic differences, engraftment and survival were similar to that observed for autologous transplants. The T cell depletion of marrow in such MHC-matched allogeneic recipients was associated in one animal, however, with early reconstitution by cells of autologous origin.     

Fetal pancreas transplantation for reversal of streptozotocin-induced diabetes in rats.

Streptozotocin-induced diabetes in rats was completely reversed by transplantation of syngeneic fetal pancreases placed beneath the kidney capsule. To accomplish complete reversal of diabetes, four or more pancreases were necessary; three resulted in partial reversal, and two produced a slight but significant effect in some recipients. Removal of the transplants resulted in the prompt return of diabetes. The islets of Langerhans in the transplants functioned homeostatically; this was indicated by regular normal blood glucose values, in addition to normal findings in blood IRI response and glucose disappearance rate after glucose injection. Disappearance of exocrine elements, with only ducts and fibrous tissue remaining, resulted in a pure endocrine organ. The advantages of this technie, such as ease of accessibility for placement, observation, and removal, are of great importance for possible application to humans.     

Vascularized islet-cell transplantation in miniature swine. I. Preparation of vascularized islet kidneys

BACKGROUND: Whereas clinical pancreatic transplantation has been highly successful in correcting the hyperglycemia of insulin-dependent diabetes mellitus (type 1), the results of islet transplantation have been disappointing. This discrepancy may be because of, at least in part, nonspecific loss of islets during the time required for revascularization. To test this hypothesis, we have designed composite kidney grafts containing vascularized autologous islets that can be used to compare the engraftment potential of vascularized versus nonvascularized islet tissue. METHODS: (1) Islet-cell isolation: miniature swine underwent either partial pancreatectomy to isolate autologous islets or total pancreatectomy to isolate minor antigen-mismatched islets. Islets were purified from excised pancreatic tissue by enzymatic digestion and discontinuous density gradient purification. Isolated islets were cultured for 3 days before transplant. (2) Creation of vascularized islet kidneys (IK): autologous islets alone (n=6), minor-mismatched islets alone (n=3), and minor-mismatched islets plus simultaneous autologous thymic tissue (n=3) were transplanted beneath the renal capsule of juvenile miniature swine. Minor antigen-mismatched islets were also transplanted into both the vascularized thymic graft of a thymokidney (to produce a thymo-islet kidney [TIK]) and the contralateral native kidney (n=3) and both the host thymus and beneath the renal capsule (n=2). All recipients receiving minor-mismatched islets were treated with a 12-day intravenous (IV) course of either cyclosporine A (CsA) at 10 mg/kg per day or FK506 at 0.15 mg/kg per day. (3) Assessment of Function: to evaluate the function of the transplanted islets, three animals bearing TIK and IK underwent total pancreatectomy 3 months following islet transplantation. RESULTS: (1) Islet-cell yields: an average of 254,960+/-51,879 (4,452+/-932 islet equivalents [IEQ]/gram of pancreas) and 374,410+/-9,548 (4,183+/-721 IEQ/gram of pancreas) viable islets were obtained by partial pancreatectomy and complete pancreatectomy, respectively. (2) Creation of IK: autologous islets engrafted indefinitely, whereas recipients of minor-mismatched islets alone rejected the islets within 2 months. However, when minor-mismatched islets were implanted into both the thymokidney and the contralateral kidney of animals bearing a thymokidney, the islets engrafted indefinitely in both sites (>3 months). Simultaneous implantation of islets into the host thymus and under the renal capsule also led to permanent engraftment of minor-mismatched islets. (3) Function of vascularized islets: three animals with both a TIK and an IK in place for 3 months underwent total pancreatectomy. All three animals maintained normoglycemia thereafter. In two of these animals, the IKs were removed 2 months after the pancreatectomy, and in both cases normoglycemia was maintained thereafter by the TIK. CONCLUSIONS: The implantation of islets beneath the autologous renal capsule permitted the establishment of a vascular supply and thereby supported normal islet-cell growth and function. The presence of thymic tissue beneath the autologous renal capsule facilitated the engraftment of minor-mismatched islets, and such grafts achieved results similar to autologous islet transplants. Therefore, the ability to create vascularized islet grafts may provide a strategy for successful islet transplantation across allogeneic and potentially across xenogeneic barriers.     

Surgical treatment of diabetes mellitus with pancreas transplantation.

OBJECTIVE. The authors compared results and morbidity in insulin-dependent diabetes mellitus (IDDM) patients undergoing preemptive pancreas transplantation (PTx) either before dialysis or before the need for a kidney transplant with IDDM patients undergoing conventional combined pancreas-kidney transplantation (PKT) after the initiation of dialysis therapy. SUMMARY BACKGROUND DATA. Combined PKT has become accepted generally as the best treatment option in carefully selected IDDM patients who either are dependent on dialysis or for whom dialysis is imminent. With improving results, the timing of PKT relative to the degree of nephropathy is evolving. However, it is not well established that the advantages of preemptive PTx can be achieved without incurring a detrimental effect on graft function or survival. METHODS. Over a 4-year study period, data on the following 3 recipient groups were collected prospectively and analyzed retrospectively: 1) 38 IDDM patients undergoing combined PKT while on dialysis (PKT:D); 2) 44 IDDM patients undergoing preemptive PKT before dialysis (PKT:ND); and 3) 20 IDDM patients undergoing solitary PTx. All patients underwent whole organ PTx with bladder drainage and were treated with quadruple immunosuppression. RESULTS. Actuarial 1-year patient survival is 100%, 98%, and 93%, respectively. One-year actuarial PTx survival (insulin-independence) is 92%, 95%, and 78%, respectively. The incidence of rejection, infection, operative complications, readmissions, and total hospital days was similar in the three groups. Long-term renal and pancreas allograft function and quality of life were similarly comparable. Rehabilitation potential favored the solitary PTx and PKT:ND groups. CONCLUSIONS. Preemptive PKT or solitary PTx performed earlier in the course of diabetes is associated with good results, facilitated rehabilitation, and may prevent further diabetic complications.     

Thymic transplantation in miniature swine. I. Development and function of the "thymokidney"

BACKGROUND: Previous studies in our laboratory have demonstrated the importance of the thymus for rapid and stable tolerance induction in an allotransplant model. The focus of the present study was to explore the feasibility of autologous thymic transplantation to produce a new transplantable organ (thymokidney) and to examine the function of subsequent vascularized thymokidney transplants in T cell development. MATERIALS AND METHODS: Eight juvenile swine received autologous thymic grafts under the renal capsule. Thymic tissue was obtained through a partial (n=6) or complete (n=2) thymectomy, and growth of the autologous thymic graft was compared between partially and completely thymectomized animals. Two of the partially thymectomized animals received irradiated (1000 cGy) as well as non-irradiated autologous thymic grafts. Graft survival, growth and evidence of thymocyte development was determined by (a) macroscopic examination of the implanted tissue, (b) histological examination, and (c) flow cytometry. Naive CD4 SP T cells were identified by CD45RA-expression. RESULTS: Growth of transplanted thymic tissue was demonstrated in all thymic graft recipients. No difference was seen between partially and completely thymectomized animals. By POD 60, the thymic grafts exhibited normal macroscopic and microscopic structure, and normal thymocyte composition. Irradiated thymic tissue displayed a similar pattern of development, but growth was markedly delayed. To evaluate thymic function of the graft, a composite thymokidney was transplanted into a recipient which had previously been thymectomized, had few circulating CD4-single positive cells and had lost MLR reactivity. The number of CD4+/CD45RA+ cells in this animal increased steadily from POD 30 to POD 150, indicating that the thymus of the composite thymokidney allograft was functional; in addition, MLR assays demonstrated that the recipient recovered immunocompetence. CONCLUSIONS: The establishment of a thymokidney by thymic autografting to the renal subcapsular space results in normal thymic growth and function, and may provide a valuable tool for studying the role of the thymus in tolerance induction. As far as we are aware, we provide the first evidence of functional vascularized thymic graft reconstituting T cells and leading to a return of a immunocompetence in a large animal model.     

Induction of kidney transplantation tolerance across major histocompatibility complex barriers by bone marrow transplantation in miniature swine

Previous studies from our laboratory have shown that permanent lymphohematopoietic chimerism can be induced in MHC-disparate miniature swine by bone marrow transplantation after lethal total-body irradiation. The purpose of the present study was to determine in this large animal model whether such chimerism would lead to permanent tolerance to a vascularized allograft without a requirement for exogenous immunosuppression. Eight miniature swine that had received MHC-mismatched BMT more than five months earlier underwent kidney transplantation (KTx) from a donor MHC matched (n = 5) or MHC mismatched (n = 3) with the BMT donor. All animals had regained in vitro responsiveness to third-party MHC antigens, as measured by mixed lymphocyte reaction (MLR), before KTx but remained nonresponsive to MHC antigens of the BMT donor and self. All three animals that received KTx mismatched for BMT donor MHC rejected promptly (mean survival time 7.0 days). Of the five animals that received KTx matched for BMT donor MHC, four showed no evidence of rejection and have functioning KTx greater than 200 days after KTx. The fifth animal had excellent renal function for 60 days but then developed a slowly rising BUN and serum creatinine, and died 75 days after KTx. The course of this animal's rejection is consistent with that previously described for rejection due to minor antigen disparities. The difference in survival of KTx matched or mismatched for the MHC of the BMT donor was statistically significant (P = 0.0062). The survival of KTx matched for the MHC of the BMT donor was significantly different from that of control animals without BMT receiving KTx mismatched for MHC (P = 0.0018). We therefore conclude that BMT is an effective means for induction of tolerance to an MHC mismatched KTx in this large animal model.     

Thymic transplantation in miniature swine: III. Induction of tolerance by transplantation of composite thymokidneys across fully major histocompatibility complex-mismatched barriers

BACKGROUND: This study determines whether composite thymokidney (TK) grafts, created by implantation of autologous thymic tissue beneath the donor's renal capsule before transplantation, could induce allogeneic transplantation tolerance across two-haplotype fully major histocompatibility complex (MHC)- mismatched barriers in juvenile MGH-miniature swine. METHODS: TK grafts were prepared by implanting autologous thymic tissue under the renal capsule of donor animals 2 to 3 months before transplantation. Four recipients were treated with a T-cell-depleting immunotoxin and received fully MHC-mismatched TK grafts plus a 12-day course of cyclosporine A (CsA). Control animals were treated with CsA alone or both CsA and immunotoxin, but with a normal kidney or a kidney implanted with autologous lymph node rather than thymus. Renal graft function was assessed by plasma creatinine levels and histologic analyses. Immunologic status was monitored by cell-mediated lympholysis assays. RESULTS: All four recipients of fully MHC-mismatched TK transplants treated with immunotoxin and a 12-day course of CsA accepted their composite renal allografts long-term. All control recipients receiving a TK and CsA alone, a normal kidney or a composite kidney containing lymph node tissue acutely rejected their grafts. CONCLUSIONS: To our knowledge, this is the first demonstration that functional vascularized thymic grafts can induce transplantation tolerance across fully MHC-mismatched barriers in a large animal model.     

外周血淋巴细胞穿孔素的表达及在肾移植排斥与耐受中的临床意义

目的 测定细胞内穿孔素（Ｐ＋）及细胞表面怕的表达,探讨与移植肾急性排斥和长期存活的关系。方法 用流式细胞仪测定２２例口才外周血细胞毒Ｔ淋巴细胞和自然杀伤细胞亚群穿孔素的表达。结果 排斥组淋巴细胞穿孔素的表达高于非排斥组,排瓜反应与ＣＤ４＾＋Ｐ＾＋下调和ＣＤ８＾＋Ｐ＾＋上调有关。冲击治疗可引起Ｐ＾＋Ｔ细胞亚群和ＣＤ５６＾＋ＮＫ细胞的降低。ＣＤ４＾＋Ｐ＾＋及ＣＤ８＾＋Ｐ＾＋细胞的下调和ＣＤ５６＾＋Ｐ上     

Bone marrow transplantation in miniature swine. III. Graft-versus-host disease and the effect of T cell depletion of marrow

Graft-versus-host disease (GVHD) has been evaluated in partially inbred miniature swine in order to study this complication of allogeneic bone marrow transplantation (BMT) in a major histocompatibility complex (MHC) genetically defined large animal model. Bone marrow from MHC homozygous ("parental") swine was injected into irradiated (900 rads total-body irradiation) MHC heterozygous ("F1") swine that shared one haplotype with the donor. All 18 animals successfully engrafted with donor bone marrow, and 17 of these developed skin rash of varying intensity depending on the extent of T cell depletion of infused marrow. Of 18 animals, 8 received undepleted bone marrow from exsanguinated donors and 2 also received additional peripheral blood lymphocytes (PBL) as a source of mature T cells. All 8 showed a moderate-to-severe rash, and the 2 pigs that received additional donor PBL developed the most severe rash. The cutaneous eruption seen in this model clinically, histologically, and immunologically resembled human GVHD. Two protocols of T cell depletion of donor bone marrow by antiporcine T cell monoclonal antibodies plus complement were tested for their effect on development of GVHD. The combination of two monoclonal antibodies, 74-12-4 (PT4) and 76-2-11 (PT8), had a marginal effect on the subsequent development of cutaneous manifestations of GVHD. However, treatment of the donor marrow by a combination of three monoclonal antibodies--PT4, PT8, and MSA4 (PT11)--effectively decreased the severity of the GVHD skin rash. These results indicate that (1) the GVHD associated with allogeneic bone marrow transplantation in swine is dependent on T cells in the marrow; (2) effective T cell depletion of donor marrow by monoclonal antibodies and complement does not prevent engraftment; and (3) this swine GVHD model, which allows study with F1 and homozygous parental combinations in an MHC genetically defined large animal, is particularly useful for the understanding of GVHD pathogenesis, prevention, and treatment.     

Proliferation rates of lymphocytes in subcutaneously transposed spleen and peripheral blood after heterotopic heart transplantation in rats

In a transplantation model using Dark Agouti rats as heart donors and Lewis rats as recipients, mean graft survival time was 7.1 days in GII, receiving no immunosuppression, and 31.4 days in GIII, animals immunosuppressed by DSG. A higher percentage of Lb in the spleen than in the PB in the transplanted groups was detected on certain days. HCT sharply decreased in immunosuppressed animals, thus suggesting a reversible suppression of erythropoesis induced by DSG.     

Bone marrow transplantation in miniature swine. II. Effect of selective genetic differences on marrow engraftment and recipient survival

In order to study the effect of defined genetic differences on bone marrow transplantation in miniature swine, five different combinations of major histocompatibility complex (MHC)-matched and mismatched bone marrow transplants were performed. Eight of nine fully MHC-mismatched allogeneic bone marrow transplants failed to reconstitute, and one animal reconstituted briefly but then died quickly thereafter. Five of six class I-matched/class II-mismatched (g----c) bone marrow transplants engrafted, showed a skin rash typical of graft-versus-host (GVH) reaction, and died 3 weeks after the marrow transplantation. None of five class II-matched/class I-mismatched (g----d) transplants engrafted. Parental marrow transplants into F1 hosts engrafted and caused GVH skin rash, with survivals from 1 to 9 months (n = 5). Serologic typing of the F1 recipients of parental marrow showed only donor-type peripheral blood lymphocytes (PBL), suggesting complete marrow replacement. Conversely, F1 into parental marrow transplants showed no engraftment (n = 6). These results indicate that resistance to MHC-mismatched allogeneic bone marrow engraftment in swine represents a host response recognizing donor class I MHC differences. This response appears to interfere with engraftment of donor bone marrow cells despite host preparation with 900-1100 rads total-body irradiation. In the absence of donor MHC class I differences, engraftment was seen despite the existence of multiple non-MHC differences, and even in the presence of class II differences. Such engraftment also led to GVH, varying in intensity according to the strength of genetic disparity (i.e., worst in parent----F1 combination). These results suggest that miniature swine should provide an effective model for study of both GVH elimination (in the parent----F1 combination) and problems of engraftment (in the F1----parent combination), the two most important obstacles to clinical allogeneic transplantation.     

Mechanism of tolerance following class I--disparate renal allografts in miniature swine. Cellular responses of tolerant animals

Previous studies utilizing a recombinant MHC haplo-type in our partially inbred miniature swine herd have demonstrated that some recipients matched only for SLA class II show long-term acceptance of renal allografts without exogenous immunosuppression. Such animals have been shown to develop systemic tolerance as evidenced by prolonged rejection times of subsequent donor-specific, but not third-party, skin grafts. In the present studies in vitro cellular responses of long-term tolerant animals and of 7 animals studied sequentially are presented. Long-term tolerant animals demonstrated responses consistent with the absence of the class I reactive helper populations normally present in naive controls. Animals studied sequentially segregated into two groups based on cellular reactivity and survival. All animals showed complete loss of antidonor class I cell-mediated lymphocytolytic (CML) reactivity by postoperative day 10. However, animals surviving less than 20 days maintained CML reactivity to donor class I plus third-party class II in the posttransplant period, while animals surviving greater than 40 days lost such reactivity. Addition of exogenous interleukin 2 could not reverse this loss. These studies suggest that tolerance induction to a renal allograft across a class I only difference involves effects on both helper and killer class I reactive cell populations.     

Increased apoptosis of peripheral blood lymphocytes in children with nephrotic syndrome

Nephrotic syndrome is accompanied by and probably related to abnormal T-lymphocyte function. Decreased stimulation of survival factors and increased levels of ”dead signals” may lead to the malfunction of many cells, including lymphocytes. In our study, we investigated the process of apoptosis within T cells in children with a first attack of nephrotic syndrome (NS). We found that the number of apoptotic T cells is greater in these patients than in both children in remission from NS and in controls. The percentage of annexin-V-fluorescein isothiocyanate (FITC)-positive CD3+ cells was 27.30± 12.13% in children with a first attack of NS, 19.22± 15.16% (P=0.006) in children in remission and 16.20± 6.13% (P=0.004) in controls. The percentage of annexin-V-FITC-positive CD3+CD4+ cells was 7.35±7.72% in children with a first attack of NS, 3.80±3.75% (P=0.0001) in children in remission and 3.82±2.01% (P=0.0002) in controls. We conclude that abnormal number and function of T lymphocytes found in NS patients may be related to an increased apoptotic rate of circulating lymphocytes. Received: 30 May 2001 / Revised: 11 October 2001 / Accepted: 13 October 2001     

Expression of HLA molecules on peripheral blood lymphocytes: a useful monitoring parameter in cardiac transplantation

During the rejection process of cardiac allografts, the expression of HLA antigens increases on various graft tissues, ie, the myocardium and the interstitial structures. However, in this type of transplant there is a paucity of knowledge about HLA expression on recipient cells, such as peripheral blood mononuclear cells. In the present study expression of HLA class I and class II antigens was monitored on peripheral blood lymphocytes prior to and during a 12-month follow-up, using flow cytometry. In our series, the frequency of acute rejection episodes was greater from the fourth to the ninth month after transplantation, coinciding with a reduction in cyclosporine blood levels. At the same time, expression of HLA class I and class II antigens significantly increased among recipients suffering from more severe acute rejection episodes compared with those showing acceptance of their grafts (P < .01). In conclusion, acute rejection episodes in cardiac transplantation were associated with up-regulation of HLA molecules on recipient peripheral blood cells. Monitoring the expression of HLA molecules on peripheral blood lymphocytes may represent an easy, noninvasive practice to individualize immunosuppressive therapy.     

Simultaneous en-bloc allotransplantation of pancreas and kidney in the animal model. Comparison of separate organ and en-bloc pancreas/kidney transplantation in swine]

The high technical complication rate of pancreas transplantation requires large animal models to improve clinical transplant survival rates. The pig is a very suitable animal due to its anatomy, physiology and immunology which are similar to humans. In this study a model of en-bloc simultaneous pancreas and kidney transplantation was established which--in contrast to separate transplantation of both organs--decreases preservation time, operation time, and clamp time. Furthermore, the rates of intra- and postoperative complications were reduced compared with separate transplantation. The donor aorta (encompassing celiac axis, superior mesenteric artery, and left renal artery) is anastomosed en-bloc to the recipients aorta in a an oblique-to-side fashion. The portal vein is anastomosed end-to-side to the left common iliac vein. The exocrine pancreatic secretions are drained via duodenocystostomy to allow for monitoring of urinary amylase for rejection. The en-bloc technique is an alternative for pediatric donor organs since the risk of vascular complications is lower compared with separate implantation of the donor vessels. Based on our results in a large animal model the en-bloc technique could be used in adult uremic diabetic patients who receive a combined pancreas-kidney transplant from a pediatric cadaver donor.