成人胰岛细胞移植治疗1型糖尿病

目的 探讨新型成人胰岛细胞分离、纯化技术分离的成人胰岛细胞移植治疗1型糖尿病的安全性与有效性.方法 采用全氟化碳液(PFC)和UW液双层冷藏胰腺,Liberase酶消化,COBE 2991型专用胰岛细胞分离机分离及连续密度梯度纯化,获取高纯度与高活性的胰岛细胞.通过上腹部小切口,胃右静脉或脐静脉插管,将经短期培养的胰岛细胞经门静脉移植到11例1型糖尿病患者肝脏内.采用达利珠单抗或阿来佐单抗进行诱导,西罗莫司和他克莫司联用的方案预防排斥反应,术后观察胰岛素使用情况,监测血糖、G-肽与糖化血红蛋白水平以及肝功能、肾功能.结果 45个胰腺中,42个成功分离出胰岛细胞,其数量平均为28.5×104胰岛当量(IEQ)/个,纯度为95.7%,活率为93.2%,胰岛素释放试验刺激指数为2.43.11例1型糖尿病患者共行胰岛细胞移植20次,其中移植1次4例,2次5例,3次2例,每次移植胰岛细胞数平均为11 200 IEQ/kg.术后随访6个月至4年,6例完全撤除胰岛素,2例胰岛素用量较术前减少80%,3例减少50%.术后血糖稳定维持在正常水平,C-肽均超过0.166 nmol/L,糖化血红蛋白基本正常,肝、肾功能正常,未发生与胰岛细胞输注相关的并发症.结论 新型成人胰岛细胞分离、纯化方法可靠,采用该技术分离、纯化的成人胰岛细胞移植治疗1型糖尿病临床效果较好.     

Pancreas islet transplantation in patients with type 1 diabetes mellitus after kidney transplantation

Diabetic patients with end-stage renal disease have a high mortality rate. A combined kidney-pancreas transplant is associated with greater life expectancy. Pancreas islet transplantation is an alternative involving a lower degree of morbidity. We present two patients, of 41 and 37 years of age, with a long history of diabetes mellitus (C-peptide negative), both with a previous kidney transplant, who had been treated with 22 and 28 U of insulin/d, respectively. Both patients had frequent episodes of unawareness hypoglycemia. Pancreatic islets were infused to a total of 7809 and 19,180 IE/kg, respectively. Basal posttransplant C peptide levels were 2.9 and 1.3 ng/mL. After the implant, one patient required occasional doses of insulin, and the other patient more than 50% reduced dose. After the first implant neither patient had any episodes of unawareness hypoglycemia. HbA1c at 4 months were 6.2% and 6.9%. There were no transplant-related complications.     

Three-yr follow-up of a type 1 diabetes mellitus patient with an islet xenotransplant

In order to alleviate the shortage of human donors, the use of porcine islets of Langerhans for xenotransplantation in diabetic patients has been proposed as a solution. To overcome rejection, we have developed a procedure for protecting the islets by combining them with Sertoli cells and placing them in a novel subcutaneous device, that generates an autologous collagen covering. A type 1 diabetic woman was closely monitored for 10 months, and then transplanted in two devices with two months of difference and a third time after 22 months. Here we present a three-yr follow-up. The close monitoring induced a rapid decrease in exogenous insulin requirements, which stabilized between 19 and 28 IU/d for nine months. After transplantation, the requirements reduced further to below 6 IU/d and for some weeks she was insulin free. Glycosylated hemoglobin levels decreased concomitantly. Porcine insulin could be detected in the serum after a glucose challenge and insulin positive cells inside a removed device after two yr. No complications have arisen and no porcine endogenous retrovirus infection has been detected through PCR and RT-PCR. This case demonstrates the feasibility of using the xenotransplantation of porcine cells to alleviate metabolic complications and insulin requirements in type 1 diabetic patients.     

Improved human pancreatic islet isolation for a prospective cohort study of islet transplantation vs best medical therapy in type 1 diabetes mellitus

HYPOTHESIS: A local multiorgan donor pancreas procurement program can provide a source for optimized isolation of purified viable islets for transplantation into patients with type 1 diabetes mellitus receiving best medical therapy. DESIGN: Prospective before-after cohort study. SETTING: Tertiary referral center. PATIENTS: Glycemic control was assessed in 10 patients with diabetes-induced renal dysfunction who were enrolled in a best medical therapy program and then crossed over to islet transplantation. INTERVENTIONS: Thirty human pancreata were retrieved from local multiorgan donors and consecutively processed with intraductal collagenase perfusion, continuous digestion, and density gradient purification (group 1, n = 9) or similarly processed but impure tissue fractions cultured in vitro and then repurified to retrieve additional islets (group 2, n = 21). Islets were implanted by percutaneous portal embolization, providing more than 10 000 islet equivalents (IE) per kilogram of body weight (infusions from 1-3 donors per patient) under cover of antithymocyte globulin, sirolimus, or mycophenolate mofetil and tacrolimus. MAIN OUTCOME MEASURES: Islet yields, purity, and cell viability (caspase 3, terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine 5-triphosphate nick-end labeling stain, and insulin secretion in vitro) were compared. In patients, monitored metabolic parameters were C-peptide secretion, insulin requirements, glycemic excursion, and hemoglobin A(1c) (HbA(1c)). RESULTS: For group 1 vs group 2, no differences were observed in pancreas age (43 vs 44 years), cold storage (5 vs 4 hours), or weight (73 vs 82 g). Group 2 yielded 453 690 IE vs 214 109 IE in group 1 (P = .002). Grafts contained 50% or more endocrine cells in both groups. No difference occurred in cell viability or insulin secretion. Islets from 90% of group 2 pancreata met release criteria for transplantation. C-peptide secretion was detected in all recipients and persisted with a median follow-up to 12 months (range, 6-21 months) after full islet transplantation. Daily insulin dependence was reversed in all patients for at least 3 months. Five patients resumed small insulin doses. Compared with the best care program, all patients had improved metabolic stability. The mean +/- SE HbA(1c) level at entry into the study was 7.8% +/- 0.5%, and this decreased to 6.9% +/- 0.2% after best care (P = .38) and further to 6.2% +/- 0.2% at 6 months after transplantation (P = .002 vs entry; P = .15 vs best care; analysis of variance). CONCLUSIONS: Local pancreas donor retrieval with islet isolation and culture conditioning enabled an offer of islets for transplantation for 90% of consecutively processed pancreata. Isolated islets secreted insulin during prolonged follow-up after implantation into patients, yielding metabolic control comparable with that achieved by best medical therapy.     

Disseminated periportal fatty degeneration after allogeneic intraportal islet transplantation in a patient with type 1 diabetes mellitus: a case report

Insulin independence after islet transplantation has been significantly improved by using new steroid-free immunosuppressive protocols and increased islet mass. Only little is known about the influence on the morphology of the liver of intraportally transplanted islets. We describe a case of disseminated periportal fatty degeneration after allogeneic intraportal islet transplantation (ITx). A 35-year-old patient with type-1 diabetes mellitus who was suffering from repeated severe hypoglycemic episodes received two sequential intraportal islet grafts. Liver structure was normal before the first ITx, based upon ultrasound and magnetic resonance imaging (MRI). One week after the first ITx, ultrasound demonstrated normal liver morphology. Four months later, at the second ITx, we detected small, disseminated, and hypodense hepatic lesions (1 to 3 mm) by ultrasound, which were confirmed by MRI and interpreted to be fatty degenerations. Histologically we found focal drop-shaped fatty degenerations with signs of mild periportal chronic inflammation. These liver alterations without clinical symptoms or pathological liver function tests matched the predicted distribution of infused islets. Glucose metabolism markedly improved after the first ITx, namely 58.6% reduction of daily insulin requirements, 1.4% decrease in HbA1c, basal C-peptide of 0.8 to 1.3 ng/dl with no severe hypoglycemia. We interpreted these benign changes in liver morphology as reactions to a local hyperinsulinemia in the neighborhood of the transplanted islets. We hypothesized that a steroid-free immunosuppression with rapamycin and tacrolimus may have contributed to changes in the portal microenvironment.     

Immunosuppression and procedure-related complications in 26 patients with type 1 diabetes mellitus receiving allogeneic islet cell transplantation

BACKGROUND: The success of sirolimus and low-dose tacrolimus in islet cell transplantation has influenced many transplant centers to utilize this novel regimen. The long-term safety and tolerability of this steroid-free immunosuppressive protocol for allogeneic islet transplantation has yet to be determined. METHODS: We transplanted 26 adult patients with long standing type 1 diabetes mellitus between April 2000 and June 2004. Immunosuppression consisted of induction with daclizumab and maintenance therapy with tacrolimus and sirolimus. Adverse events (AEs) in patients were followed and graded using the Common Terminology Criteria for Adverse Events, version 3.0 (National Cancer Institute). RESULTS: To date, the majority of patients were able to remain on the immunosuppression combination for up to 22+/-11 months. Four patients were successfully converted to Mycophenolate Mofetil due to tacrolimus-related toxicity. Withdrawal from immunosuppression was decided in four patients due to hypereosinophilic syndrome, parvovirus infection, aspiration pneumonia, and severe depression, respectively. Six patients required filgrastim therapy for neutropenia. Transient elevation of liver enzymes was observed in most patients early after islet infusion. Increased LDL in 20 patients required medical treatment. CONCLUSION: There was a varying range of AEs, most of them mild and self-limiting; however, some required urgent medical attention. The majority of patients were able to tolerate and remain on this effective regimen. To date, no deaths, cytomegalovirus disease, graft-versus-host disease, or posttransplant lymphoproliferative disease has been observed.     

胰岛素依赖型糖尿病患者胚胎胰腺组织脑内移植的远期疗效观察与免疫学研究

目的观察脑内胰腺组织移植治疗胰岛素依赖型糖尿病（ＩＤＤＭ）的远期疗效,并探讨其免疫学变化。方法应用短期培养的人胎胰腺组织脑内移植,共施行３０例,移植术后随访１～７年。结果移植后１年内有效２６例,无效４例;移植后２～３年随访２１例,有效１７例,失效４例;４～５年随访１８例,有效１５例,失效３例;移植后６～７年随访１５例,有效９例,失效６例。在移植有效的病例中,随访资料较完整者２３例,有效维持时间为（４．９±１．７）年,１１例完全或间断停用胰岛素治疗,停用时间达（３．１±１．９６）年,其中超过３年者６例。移植前胰岛细胞抗体（ＩＣＡ）阳性的６例,４例移植无效;移植无效或后期复发的患者,外周血Ｔ淋巴细胞亚群明显失衡。结论脑内胰腺组织移植治疗ＩＤＤＭ的远期效果显著;ＩＣＡ阳性者不宜行胰腺组织移植     

成人胰岛移植治疗2型糖尿病三例

目的 探讨成人胰岛移植治疗2型糖尿病的安全性与有效性.方法 为3例合并慢性肾功能衰竭的2型糖尿病患者共施行4次胰岛移植,其中2例接受成人胰岛细胞和肾联合移植(每例的胰岛细胞和肾脏来自同一供者,以下简称"联合移植"),1例为肾移植术后5个月连续2次接受胰岛细胞移植,2次胰岛细胞移植间隔9 d.3例移植前均依赖胰岛素治疗,用量为0.5～0.7IU·kg-1·d-1.采用经皮肝门静脉穿刺,胰岛悬液重力输注,将移植胰岛注入到受者的肝脏内.2例联合移植者以巴利昔单抗诱导,术后采用低剂量他克莫司+西罗莫司维持治疗;1例肾移植术后胰岛移植者采用环孢素A+吗替麦考酚酯维持治疗.术后观察胰岛素使用情况,监测血糖、C-肽与糖化血红蛋白水平.结果 第1例联合移植者术后第3天血糖正常,停用外源性胰岛素,但术后1个月时患者的血糖逐渐升高,遂给予少量胰岛素,与术前相比,胰岛素减量2/3.第2例联合移植者术后24 h血糖即恢复正常,停用外源性胰岛素,术后第5天死于肝穿刺部位出血及继发感染.肾移植术后胰岛移植的患者在第1次胰岛移植后胰岛素用量即减少1/2,第2次胰岛移植后完全停用胰岛素.与术前相比,长期存活的2例受者术后空腹C-肽和餐后2 h C-肽值平均升高600 pmol/L.联合移植者术后糖化血红蛋白波动于6.7%～7.3%,肾移植后胰岛移植受者的糖化血红蛋白波动于5.5%～5.9%.结论 成人胰岛移植可有效治疗达到胰岛素依赖期的2型糖尿病.

Abstract：

Objective To evaluate the effect of islet transplantation for patients with type 2diabetes mellitus (DM). Methods Since December 2007, 4 cases of islet transplantations were performed on 3 patients with type 2 DM and end-stage renal disease (ESRD). Two patients received simultaneous islet-kidney transplant from single-donor (SIK), and one received 2 consecutive islet transplants 5 months following kidney transplantion (IAK). All recipients given insulin with a dose of percutaneous transhepatic portal catheterization. Anti-CD25 monoclonal antibody was used as induction. For SIK, low-doses of Tacrolimus and sirolimus were used as maintenance immunosuppression protocol. For IAK, the maintenance protocol included cyclosporine and MMF.Insulin dose, the level of blood glucose, C-peptide and the value of HbA1 were observed. Results The first patient of SIKhad normal glucose level 3 days after surgery and became insulin independent within the first month, but insulin was administered gradually and the dose reduced to 1/3. The second patient of SIK died of bleeding and secondary infection of liver puncture site 5 days following operation, the blood glucose level recovered to normal 24 h after operation. The insulin dose of the patient of IAK was reduced to 1/2 after the first transplant. The patient became insulin free after the second operation. The level of fasting and postprandial C-peptide of the surviving recipients increased by 600 pmol/L. The value of HbA1 of the SIK was 6.7 %～7.3 %, while that of the IAK was 5. 5 %～ 5. 9 %. Conclusion Islet transplantation is an effective treatment for patients with type 2 DM.     

Autoantibody response to islet transplantation in type 1 diabetes.

Islet allotransplantation into patients with autoimmune type 1 diabetes represents a reexposure to autoantigen. Here, measurement of antibodies to GAD and IA-2 autoantigens before and after islet transplantation in 36 patients (33 receiving islet plus kidney grafts with cyclosporin and steroid-based immunosuppression, and 3 receiving solitary islet transplants with mycophenolate but cyclosporin-free immunosuppression) demonstrated marked rises in GAD antibodies within 7 days posttransplantation in 5 patients (3 receiving islet after kidney transplants, and 2 receiving solitary islet transplants) and within 30 days in the third patient receiving solitary islet transplantation. GAD antibodies were of the IgG1 subclass, against major autoantigenic epitopes, and in cases of islet after kidney transplants, the responses were short-lived and not accompanied by HLA antibodies. Two of these patients had subsequent marked rises of IA-2 antibodies, and an additional patient had a marked rise in IgM-GAD antibodies 3 years after transplantation. Insulin independence was not achieved in patients with autoantibody elevations and was significantly less frequent in these patients. These data are consistent with a reactivation of autoimmunity that may be dependent on immunosuppression therapy and is associated with impaired graft function.     

Optimized parameters for microencapsulation of pancreatic islet cells: an in vitro study clueing on islet graft immunoprotection in type 1 diabetes mellitus

Alginate (AG)-based microcapsules may provide a selective permeable and biocompatible physical barrier to prevent islet graft (TX)-directed immune destruction. However, extent of the achieved immunoprotection will continue to be variable and unpredictable until the role of the individual mechanisms involved with TX-related inflammatory cell and immune reactivity are clarified. Macrophages (M) are believed to play a pivotal role in controlling the host/TX interaction and its consequences. We then have studied the effects of isolated rat M and their secretory products on allogeneic islets enveloped in variably sized and configured microcapsules, within in vitro mixed islet-M cocultures. In particular, we aimed to determine the sequence of immune or not immune specific cascade of early events that derive from such on interaction. One of the specific aims was to assess whether the membrane's physical intactness and conversely its even minimal rupture, along with the microcapsules' size (i.e., large vs. small) would significantly impact M reactivity and, thereby, the encapsulated islet viability and function. Special care was taken to evaluate extent of the elicited reactivity by meticulously monitoring cytokine, N2 derivative, and other proinflammatory protein curve profiles during the early M activation process. The study has preliminarily shown that, for equally formulated microcapsules, the capsular size and membrane's morphologic thoroughness are key to prevent M reactivity and possibly avoid the intracapsular islet cell damage. While elucidation of pathways involved with the encapsulated islet TX-directed host's responsiveness actually is in progress, it has clearly emerged that microcapsules should comply with well-defined physical properties and formulation specifications in order to obviate the primum movens of the inflammatory reaction process.     

Encephalomyocarditis virus-induced diabetes mellitus treated by islet transplantation.

There is considerable evidence that at least some cases of juvenile onset diabetes mellitus in humans are a result of viral infection. Viral-induced diabetes in mice may provide an experimental counterpart more similar to the clinical situation than chemical-induced diabetes. Our experiments in such mice indicate that islet transplantation is effective in ameliorating viral-induced diabetes and is encouraging for ultimate clinical application of islet transplantation to juvenile onset insulin-dependent diabetics. In addition, our results show that islets in ectopic sites outside of the pancreas are resistant to damage induced by primary viral infection. The mechanism of this resistance is obscure and will be the subject of future investigations.     

胰岛移植治疗1型糖尿病的现状和未来

1型糖尿病是一种全球性的慢性自身免疫疾病,会造成机体一系列代谢紊乱综合征,严重危害人类健康。胰岛移植可以通过增加1型糖尿病患者体内胰岛细胞的数量,减少患者对外源性胰岛素的依赖、有效控制血糖以及防治长期并发症,是一种安全可靠的治疗方法,这都是外源性胰岛素治疗和移植风险极高的全胰腺移植无法实现的。本文就胰岛移植供受体选择、胰岛细胞的分离纯化及胰岛移植、胰岛移植后的免疫抑制治疗、胰岛移植后的监测指标、胰岛移植临床应用的现状以及面临的挑战做一综述。     

Reluctance of French patients with type 1 diabetes to undergo pig pancreatic islet xenotransplantation

INTRODUCTION: Type 1 diabetes could possibly be treated by transplantation of pig pancreatic islets. In addition to medical difficulties and ethical problems, social hurdles may need to be overcome. We have evaluated the attitude of patients with type 1 diabetes to the xenotransplantation of pig pancreatic islets and to the potential risks associated with such treatment. METHODS: A survey of 214 patients with type 1 diabetes was carried out in France based on a multiple-choice questionnaire. RESULTS: At first, 52.0% of these patients indicated that they would agree to receive pig islet xenografts. The main sources of reluctance were the 'risk of disease transmission' (55.5%) and 'risks not yet identified' (48.7%). After they were told of the risk of cancer or infection associated with immunosuppression, 74.9% of the respondents chose to refuse the transplant, compared with 48.0% before they heard of such risks. A 68.1% would refuse the xenotransplant if it would not exempt them completely from being treated by insulin injections. Discontinuing insulin injections was the most important priority for diabetic patients (73.5%), rather than limitation of diabetes-related complications (52.5%) or increase in life expectancy (44.0%). After they were informed of all of the risks associated with the procedure, 70.5% of the respondents decided they would rather not take any risks, and said they would refuse pig islet transplantation. CONCLUSION: When diabetic patients learned about potential infectious risks and other risks associated with immunosuppression, reluctance to undergo xenotransplantation gained in significance or even led to refusal of the procedure.     

New mouse model to study islet transplantation in insulin-dependent diabetes mellitus

BACKGROUND: Islet transplantation studies with diabetic rodents frequently use treatment with diabetogens such as alloxan or streptozotocin to render hosts hyperglycemic. These chemicals produce unwanted toxic side effects, which complicate interpretations of damage produced by hyperglycemia versus direct toxin-induced damage. A mouse that spontaneously developed insulin-sensitive diabetes without beta-cell autoimmunity would provide an excellent vehicle for testing beta-cell replacement protocols. The Ins2Akita mutation disrupts normal insulin processing and causes a failure in secretion of mature insulins, which results in the early development of hyperglycemia. This report examines the insulin sensitivity of mice that carry Ins2Akita and their responsiveness to engraftment with syngeneic pancreatic islets. METHODS: Ten-week-old C57BL/6J-Ins2Akita/+ males were given 1 unit of insulin to determine insulin sensitivity. Also, 10-week-old, hyperglycemic B6-Ins2Akita/+ received either 400 islets isolated from syngeneic C57BL/6J males (n=7) or from allogeneic BALB/cJ males (n=5) under the renal capsule. These mice were followed for 8 weeks after engraftment or until remission of euglycemia. Nephrectomy of the graft-containing kidney was performed on mice that remained euglycemic. These mice were then followed for 2 weeks for return of hyperglycemia. RESULTS: B6-Ins2Akita/+ mice are insulin responsive. Insulin treatment of hyperglycemic B6-Ins2Akita/+ males significantly lowered blood glucose values within 1 hr. In addition, B6-Ins2Akita/+ recipients of syngeneic islet grafts reversed their diabetic state in less than 72 hr. These islet-engrafted mice remained normoglycemic until removal of the graft-containing kidney. Removal of the graft resulted in a return to hyperglycemia. Mice that received allogeneic grafts efficiently rejected the graft. CONCLUSIONS: Our data support the hypothesis that B6-Ins2Akita/+ mice are insulin sensitive and provide an excellent model for islet transplantation studies. In addition, the reduced beta-cell mass and the absence of beta-cell autoimmunity, coupled to the fact that these mice also reject allografts, suggest that these mice may be useful for a variety of other applications, including testing functionality of human islets prepared for transplantation and perhaps also for exploring beta-cell restorative therapy using pancreatic islet stem cells.     

Noninvasive diagnosis of recurrent autoimmune type 1 diabetes after islet cell transplantation

Islet cell transplantation is curative therapy for patients with complicated autoimmune type 1 diabetes (T1D). We report the diagnostic potential of circulating transplant islet–specific exosomes to noninvasively distinguish pancreatic β cell injury secondary to recurrent autoimmunity vs immunologic rejection. A T1D patient with hypoglycemic unawareness underwent islet transplantation and maintained normoglycemia until posttransplant day 1098 before requiring exogenous insulin. Plasma analysis showed decreased donor islet exosome quantities on day 1001, before hyperglycemia onset. This drop in islet exosome quantity signified islet injury, but did not distinguish injury type. However, analysis of purified transplant islet exosome cargoes showed decrease in insulin‐containing exosomes, but not glucagon‐containing exosomes, indicating selective destruction of transplanted β cells secondary to recurrent T1D autoimmunity. Furthermore, donor islet exosome cargo analysis showed time‐specific increase in islet autoantigen, glutamic acid decarboxylase 65 (GAD65), implicated in T1D autoimmunity. Time‐matched analysis of plasma transplant islet exosomes in 3 control subjects undergoing islet cell transplantation failed to show changes in islet exosome quantities or intraexosomal cargo expression of insulin, glucagon, and GAD65. This is the first report of noninvasive diagnosis of recurrent autoimmunity after islet cell transplantation, suggesting that transplant tissue exosome platform may serve as a biomarker in islet transplant diagnostics.     

Acute postoperative mononeuropathy in a patient with type II diabetes mellitus]

Diabetes mellitus is an endocrinologic illness that affects multiple systems. One of then is the nervous system. It has been attempted to establish a relationship between metabolic abnormalities associated with diabetes and the degree of nervous system affectation. We present a case of mononeuritis involving the left peroneal nerve which occurred suddenly during the postoperative phase of a thoracoplasty in a patient with type II diabetes mellitus. The clinical picture was associated with a hyperglycemic nonketotic decompensation. We analyze the precipitating factors and their possible prevention.