Skin allograft survival following intrathymic injection of donor bone marrow.

BACKGROUND: Success has been reported using intrathymic injection in the preconditioning regimen to induce allograft tolerance. Although long-term stable tolerance has been achieved in numerous rodent vascularized solid organ allograft models, tolerance to skin transplants has only been achieved across minor antigenic or concordant species disparities. This study sought to induce tolerance across an allogeneic barrier in a rat model with a major genetic disparity. MATERIALS AND METHODS: Lewis rats were injected intrathymically with 1 x 10(8) Brown-Norway (BN) bone marrow cells and intraperitoneally with 1.0 cc of rabbit anti-rat anti-lymphocyte serum (ALS). Twenty-one days later, BN skin grafts were placed on the injected animals. Control groups were included to isolate the effect of technique, thymic manipulation, strain specificity, and ALS. RESULTS: Animals receiving both intrathymic bone marrow cells and ALS had a skin graft median survival time of 24 days versus 8 days for the control group (P = 0.003). Groups receiving anti-lymphocyte serum alone or intrathymic bone marrow cell injection alone exhibited no skin graft survival prolongation. Mixed lymphocyte reactions revealed normal responsiveness of tolerant animal lymphocytes to donor strain lymphocytes. CONCLUSION: This protocol utilizing the intrathymic injection of donor bone marrow cells along with short-term immunosuppression with anti-lymphocyte serum produced markedly prolonged survival of skin allografts transplanted across a major histocompatibility barrier. Although tolerance was incomplete, significant prolongation has not previously been reported in genetic disparities of this degree. These results suggest that the application of this technique for central immune modulation may be beneficial for allograft tolerance induction and deserves further study in large animals models.     

Prolonged improvement of total pancreatic allograft function by previous intrathymic bone marrow cell injection in rats

Donor-specific induction of tolerance was previously achieved in the diabetic rat by intrathymic injection of pancreatic islets. It allowed a secondary islet graft in any site without immunosuppression. Since total pancreatic graft in man is metabolically more proficient than islet graft, we attempted tolerance induction for total vascularized pancreas transplantation in diabetic BN recipient rats by an intrathymic bone marrow cell (BMC) injection from Lewis donor rats, associated to an antilymphocyte antibody (ALS) administration. Control groups consisted of isogenic grafts, allogenic grafts without tolerance induction and allogenic grafts with ALS alone. In all grafted groups, mean blood glucose and plasma insulin were normalised within 24 h. Graft rejection (clinically suggested by diabetes recurrence and later confirmed by histology) appeared at 18 +/- 2 postoperative days in the absence of intrathymic BMC injection and at 36 +/- 8 days in the group with BMC injection (p < 0.05). Intrathymic bone marrow graft was successful in delaying rejection in our study.     

胸腺内胰岛移植的实验研究

目的观察胸腺内和肾包膜下胰岛移植对移植物存活时间的影响。方法以Ｃ５７ＢＬ／６小鼠为受体,ＢＡＬＢ／ｃ小鼠为供体,肾包膜下胰岛移植分为单纯移植和移植的同时加腹腔内一次性注射兔抗小鼠胸腺细胞血清（ＡＴＳ）两组,胸腺内移植组亦分为单纯移植和移植加用ＡＴＳ两组。结果单纯胸腺内移植组其移植物的存活期为（１９．５±１０．１）天,长于单纯肾包膜下移植组（１４．０±２．１）天;移植的同时加用ＡＴＳ,则肾包膜下移植组移植物的存活期为（４３．０±１１．７）天,胸腺内移植组可达（９３．８±２５．５）天以上,其中６只（６／８）胸腺内移植物获长期存活,并且诱导了受体对供体的特异无反应性。结论胸腺可能为胰岛移植的理想部位,而且在诱导免疫耐受中具有重要作用。     

Prolongation of islet allograft survival.

Pretreatment of donor rats with irradiation and silica followed by in vitro culture of the islets for 1 to 2 days prolonged survival of allografts across a minor histocompatibility barrier if "hand-picked," clean islets were used for transplantation. Pretreatment of donor rats with irradiation and silica in conjunction with a single injection of antilymphocyte serum (ALS) into the recipient produced a prolongation of survival of hand-picked islets transplanted across a major histocompatibility barrier.     

Evaluation of intrathymic islet transplantation in the prediabetic period.

BACKGROUND. Beta-cell destruction in type I diabetes mellitus results from a chronic autoimmune process. Exposure of thymic T cells to islet antigens during the prehyperglycemic phase of diabetes may alter the likelihood of autoimmune damage to beta cells in the native pancreas. Thus we evaluated whether prophylactic major histocompatibility complex (MHC)-incompatible intrathymic islet allografts could prevent hyperglycemia and native pancreatic beta-cell destruction. METHODS. At 4 to 6 weeks of age, diabetes-prone BioBreeding rats received intrathymic injection of 1500 to 2000 noncultured MHC-incompatible Lewis islets. No immunosuppression was administered. Age-matched littermates underwent intrathymic injection of saline solution. RESULTS. None of 13 BioBreeding rat recipients of prophylactic intrathymic Lewis islet allografts became hyperglycemic versus 13 of 13 control rats (p less than 0.001). The age at onset of diabetes in the control group ranged from 77 to 104 days (mean, 86 days). Normoglycemia in recipients of intrathymic islet allografts persisted for greater than 8 months after transplantation, and thymectomy (graft removal) did not precipitate hyperglycemia. CONCLUSIONS. Prophylactic intrathymic MHC-incompatible islet allografts effectively prevent hyperglycemia and native beta-cell destruction in an animal model of autoimmune diabetes. Rejection and autoimmune destruction of intrathymic MHC-incompatible islet allografts were not seen after transplantation in the prediabetic (prehyperglycemic) period. Intrathymic islet allografts at an early age (before puberty) preserve native beta-cell function and may prevent or retard thymic atrophy.     

A new method of bone marrow transplantation leads to extension of skin allograft survival

Tolerance induction through allogeneic bone marrow transplantation is an alternative method to chronic immunosuppression in maintaining long-term allograft survival. In this article, we introduce a new method of bone marrow allotransplantation, which preserves its natural microenvironment and does not require marrow processing or recipient conditioning. A total of 43 skin graft transplantations were performed in nine experimental groups between isogeneic [Lewis to Lewis (LEW, RT1(1))] and allogeneic [Lewis x Brown Norway (LBN --> F1, RT1(1+n)) to Lewis] rats under 35-day protocol of alphabeta T-cell receptor (TCR) monoclonal antibody (mAb) and cyclosporine (CsA) protocol. Monotherapies combined with "crude" bone marrow transplantation resulted in extended survival up to 21 days under CsA and up to 10 days under alphabeta-TCR mAb protocol. The use of combined protocol of alphabeta-TCRmAb/CsA with crude bone marrow transplantation resulted in the extension of skin allograft survival up to 65 days (P < .05). This new simple method of "crude" bone marrow allotransplantation without recipient conditioning is a promising, minimally invasive technique with a potential for direct clinical application.     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受.方法将114只日本白色家兔和Dutch家兔分为对照组和实验组,日本白色家兔作为供体,Dutch家兔作为受体.对照组,在不使用免疫抑制剂的情况下,将12只日本白色家兔与12只Dutch家兔行相同面积的背部全厚皮肤互换移植,观察其成活时间.实验组,将45只日本白色家兔和45只Dutch家兔行全厚皮肤移植的同时行骨髓移植,然后将作为受体的Dutch家兔分为A,B,C,D四组,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植,观察移植皮肤的成活时间.结果对照组,供体与受体移植皮肤的平均成活时间分别为(12.0±1.7)天和(10.3±1.3)天.实验组,A,B,C,D四组移植皮肤的平均成活时间分别为(61.0± 7.2)、(80.7± 10.4)、(78.8± 12.7)、(88.0± 6.0)天.结论通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验,旨在为临床应用提供了理论基础及可靠依据,为同种组织重建提供一个新方法.     

骨髓移植诱导特异性免疫耐受同种皮肤移植的动物实验研究

目的　证实通过动物实验模型的骨髓移植可以诱导同种皮肤移植的免疫耐受。方法　将 114只日本白色家兔和Dutch家兔分为对照组和实验组 ,日本白色家兔作为供体 ,Dutch家兔作为受体。对照组 ,在不使用免疫抑制剂的情况下 ,将 12只日本白色家兔与 12只Dutch家兔行相同面积的背部全厚皮肤互换移植 ,观察其成活时间。实验组 ,将 4 5只日本白色家兔和 4 5只Dutch家兔行全厚皮肤移植的同时行骨髓移植 ,然后将作为受体的Dutch家兔分为A ,B ,C ,D四组 ,分别行非致死量的γ射线全身照射的骨髓细胞移植及同种皮肤移植 ,观察移植皮肤的成活时间。结果　对照组 ,供体与受体移植皮肤的平均成活时间分别为 (12 .0± 1.7)天和 (10 .3± 1.3)天。实验组 ,A ,B ,C ,D四组移植皮肤的平均成活时间分别为 (6 1.0± 7.2 )、(80 .7± 10 .4 )、(78.8± 12 .7)、(88.0± 6 .0 )天。结论　通过骨髓移植导特异性免疫耐受同种皮肤移植的动物实验 ,旨在为临床应用提供了理论基础及可靠依据 ,为同种组织重建提供一个新方法     

骨髓移植诱导免疫耐受家兔全耳同种移植

目的　为了减轻免疫诱导过程中对受体的侵袭 ,进一步探讨以家兔为动物模型 ,通过异体骨髓移植诱导同种皮肤移植免疫耐受。方法　通过骨髓腔内直接注射异体骨髓细胞 ,进行骨髓移植。结果　皮片的平均存活时间是 (88.0± 6 .0 )天 (P <0 .0 0 1 )。为了探讨其普遍性 ,我们用此诱导方法 ,还进行了同种全耳移植。平均存活时间是 (1 4 6 .0± 1 5 .1 )天 ,并有 1只移植耳存活时间已超过 1年 ,未见任何排斥反应。结论　本研究证明了以家兔为动物模型 ,在不使用免疫抑制剂的情况下 ,通过骨髓腔内直接注射异体骨髓细胞 ,进行骨髓移植的方法 ,不仅在皮肤移植上可以诱导长期稳定的特异性免疫耐受 ,而且在实质性器官耳的同种移植上取得了良好的效果     

Experimental pancreatic islet transplantation.

Transplantation of isolated islets of Langerhans has been suggested as a treatment of certain forms of diabetes mellitus. Injection of 200-400 syngeneic pancreatic islets isolated by collagenase digestion into the pancreas or submandibular gland of diabetic rats rendered most of the hosts nearly normoglycaemic. Blood glucose determinations were monitored for 2 months after islet transplantation. Although intrapancreatic and intrasubmandibular implantation reduced hyperglycaemia and polyuria in these animals, consistent normal values were rarely achieved.     

骨髓移植诱导免疫耐受家兔全耳同种移植

目的为了减轻免疫诱导过程中对受体的侵袭,进一步探讨以家兔为动物模型,通过异体骨髓移植诱导同种皮肤移植免疫耐受.方法通过骨髓腔内直接注射异体骨髓细胞,进行骨髓移植.结果皮片的平均存活时间是(88.0±6.0)天(P<0.001).为了探讨其普遍性,我们用此诱导方法,还进行了同种全耳移植.平均存活时间是(146.0±15.1)天,并有1只移植耳存活时间已超过1年,未见任何排斥反应.结论本研究证明了以家兔为动物模型,在不使用免疫抑制剂的情况下,通过骨髓腔内直接注射异体骨髓细胞,进行骨髓移植的方法,不仅在皮肤移植上可以诱导长期稳定的特异性免疫耐受,而且在实质性器官耳的同种移植上取得了良好的效果.     

Prior renal transplantation does not facilitate subsequent canine islet allograft survival

Successful islet allografts can be established in rats bearing long surviving renal allografts, without additional immunosuppression, when kidney and islet donor animals are of the same strain. The applicability of such a scheme to clinical practice has been investigated in a large animal model of diabetes: the pancreatectomized dog. Eight dogs with previously established renal allografts and immunosuppressed with cyclosporin A received islet allografts from their respective original kidney donors. The median islet graft functional survival was only 10.5 days, significantly less than for six similarly immunosuppressed dogs receiving islet allografts alone (48.5 days, P less than 0.05). Three of the sequentially transplanted dogs had had no renal graft rejection episodes before islet transplantation, yet their islet grafts were all rejected within 19 days. In the pancreatectomized dog, prior donor specific renal transplantation has an adverse effect upon subsequent islet graft survival.     

Pancreatic islet transplantation into the bone marrow of the rat

BACKGROUND: The liver is the current site for pancreatic islet transplantation, but presents important technical complications and limitations. We asked whether pancreatic islets could be engrafted in the bone marrow, an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. METHODS: We implanted pancreatic islet isografts (Lewis islets to Lewis rats), allografts (Wistar Furth islets to Sprague Dawley rats), and xenografts (Tilapia islets to Sprague Dawley rats) into the bone marrow of nondiabetic recipients and assessed survival by histology and immunocytochemistry. No immunosuppression was used. RESULTS: Isografts and allografts showed positive staining for insulin and glucagon and no evidence of allograft rejection up to 21 days posttransplant. Xenografts were acutely rejected. CONCLUSIONS: The bone marrow may be an attractive alternative site for pancreatic islet transplantation. The acceptance of allografts and isografts but rejection of xenografts suggests a selective phenomenon for the inflammatory process.     

Intrathymic islet transplantation in the canine: I. Histological and functional evidence of autologous intrathymic islet engraftment and survival in pancreatectomized recipients

BACKGROUND: Although an attractive alternative to daily insulin therapy, allogeneic pancreatic islet transplantation has yielded suboptimal results in clinical trials, in contrast to islet allotransplantation in animal models, which have demonstrated consistent success. The successful transplantation of isolated islets to the thymus, with a single concomitant dose of antilymphocyte serum, has been demonstrated in rodents, and more significantly, such intrathymic islet allografts have been shown to induce recipient tolerance toward subsequent extrathymic donor strain islet allografts. Intrathymic islet autotransplantation has been pursued, as a prelude to studies of allogeneic IT islet transplantation and tolerance induction, in canine, porcine, and non-human primate models, to assess the large animal thymus as a site capable of supporting a viable islet graft. However, little functional or histological evidence has established definitive survival of islets transplanted within the thymus of a phylogenetically advanced species, which may be requisite to tolerance induction. This study describes the successful intrathymic autotransplantation of isolated islets using a canine model. METHODS: Purpose-bred juvenile dogs, aged 4-6 months, underwent partial (n=4), or total pancreatectomy (n=11), and transplantation of autologous islets. The pancreas (or pancreatic limb) was distended with collagenase solution, and digested using a modification of the semiautomated system of Ricordi. Islets were purified by discontinuous gradient centrifugation, using Euroficoll (ficoll in Euro-Collin's kidney preservation solution). Partially pancreatectomized canines underwent IT transplantation of purified autologous islets (8000+/-4000 IEs), and were killed 8 weeks posttransplant. Totally pancreatectomized canines underwent transplantation of autologous islets to the liver (via portal vein embolization, n=5, IPO group) or the thymus (via direct IT injection, n=6, IT group), and were serially evaluated for a period of 8 weeks posttransplant to assess fasting blood glucose (FBG), serum insulin (SI) levels, and i.v. glucose tolerance (IVGTTs). K values (defined as the %-decrease/minute of the log(e) of blood glucose values) were calculated from IVGTT results. RESULTS: After autotransplantation in this cohort of animals, five of five IPO, and three of six IT islet recipients, remained normoglycemic (mean FBG< or =250 mg%) immediately posttransplant, and all recipients exhibited significantly elevated SI levels compared to apancreatic controls (n=10, followed 72 hr postpancreatectomy). Normal k values (=-1.1) were observed in two of five IPO, and in one of six IT recipients, 8 weeks after transplantation, and thymic tissue insulin content was increased compared to non-islet-bearing thymi (93.7+/-48.6 ng/g tissue vs. 0.7+/-0.4 ng/g tissue). At 8 weeks posttransplantation thymi from both partially and totally pancreatectomized animals were resected and processed for histological examination. Microscopic analysis of islet-bearing thymi revealed positive staining for islet-specific hormones (insulin and glucagon) within all IT recipients., Identification of islets within thymi of hyperglycemic IT recipients was problematic as islet beta cells were highly degranulated as a result of the recipients glycemic state. CONCLUSIONS: These results indicate that autologous islets, transplanted to the canine thymus, engraft, function, and survive for up to 8 weeks after islet autotransplantation to the canine thymus and establish the feasibility of intrathymic islet transplantation in a phylogenetically advanced animal model. The ability of islets to survive within the thymic environment for a period of at least 8 weeks after transplantation suggests that the successful induction of specific unresponsiveness secondary to intrathymic transplantation will not be impaired or limited by the inability of a viable islet mass to survive within the thymus for a sufficient period.