Successful reversal of spontaneous diabetes in dogs by intraperitoneal microencapsulated islets.

Long-term euglycemia by intraperitoneal transplantation of microencapsulated islets has not been described in the diabetic large animal model. In this study, we report the successful long-term reversal of diabetes by this method in spontaneous diabetic dogs. We have identified fundamental mechanism(s) associated with alginate-based microcapsule fibrosis, and have devised methods to ameliorate this problem. These include the use of purified alginate of low mannuronic acid content and cytokine suppression. Ten insulin-dependent, spontaneous diabetic dogs (insulin requirement 1-4 units/kg/day; absence of circulating C-peptide and diabetic K-values of 0.6 +/- 0.4) were entered into the study. Islets from mongrel donor pancreata were isolated and transplanted intraperitoneally either as free islet controls (n = 3) or as microencapsulated islet allografts (n = 7). In all seven encapsulated islet recipients, euglycemia was achieved within 24 hr (serum glucose failing from 304 +/- 117 to 116 +/- 72 mg/dl). IVGTT performed 14 days after islet transplant demonstrated normalization of K-values changing from a pretransplant level of 0.6 +/- 0.4 to 2.6 +/- 0.6. All animals receiving encapsulated islets remained euglycemic, free of the need for exogenous insulin, for a period of 63-172 days, with a median insulin-independence for 105 days. In contrast, recipients receiving free islets rejected their graft within seven days of implantation. In conclusion, this is the first report of long-term successful reversal of spontaneous diabetes in the large animal model by an intraperitoneal injection of encapsulated islets. The potential exists for this form of therapy to be explored in the treatment of type I diabetes in man.     

微囊化人胎胰岛移植治疗小鼠实验性糖尿病的观察

目的 :探讨微包囊技术在解决胰岛移植免疫排斥问题中的作用。方法 :将用链脲霉素 (STZ)制备的合格糖尿病模型鼠 2 1只随机分为 3组 ,每组 7只。空囊组腹腔内植入 5 0 0～ 6 0 0个空囊 ,游离胰岛组植入经胶原酶消化制备的人胎胰岛细胞 10 0 0± 10 0个 ,微囊组植入 10 0 0± 10 0个微囊包裹的胰岛细胞。结果 :游离胰岛组和微囊组小鼠在完全停用胰岛素的情况下 ,术后血糖分别降至 7.94± 2 .36mmol.L-1和 7.0 7± 1.15mmol.L-1,与空囊组比较差异有统计学意义 (t=13.170　P <0 .0 0 1,t=2 4 .999　P <0 .0 0 1) ,分别持续 7.4 3± 3.4 2天和 78.4± 2 1.2 7天 (t =8.6 5P <0 .0 0 1)。结论 :该微囊化人胎胰岛移植具有良好的组织相容性和免疫隔离作用 ,明显延长移植胰岛的存活时间     

Transplantation of isolated islets of Langerhans in diabetic dogs. I. Results after allogeneic intraportal islet transplantation.

Twenty partially inbred German shepherd dogs were made diabetic by intravenous administration of streptozotocin (25 mg/kg) 1 and 3 days after partial pancreatectomy. Ten diabetic dogs received intraportal transplants of allogeneic islets of Langerhans isolated by collagenase digestion and Ficon gradient separation. After transplantation the mean fasting serum glucose level fell from 368 ± 74 to 108 ± 52 mg/dl. Normal glycemia was maintained for at least 3 weeks before rejection occurred. The mean survival time of the 10 recipients was 76 ± 30 days, while the 10 diabetic control dogs had a mean survival time of 30 ± 7 days. Intravenous glucose tolerance test curves were clearly better in recipient dogs than in the diabetic control dogs for at least 4 weeks after islet transplantation. Concentrations of immunoreactive insulin (IRI) in the portal vein in the superior vena cava were also significantly higher than in the diabetic control dogs 4 weeks after islet transplantation. The mean peak concentration of IRI in the superior vena cava of transplanted dogs after glucose administration was higher than that in the portal vein, indicating that the transplanted islets secreted insulin in response to glucose stimulation. Portal hypertension or disturbance of liver function did not occur after transplantation of isolated islets. These results show that the intrahepatic site is appropriate for transplantation of isolated islets in a large animal model of diabetes, and provide a basis for future application in man.     

Prolonged reversal of diabetic state in NOD mice by xenografts of microencapsulated rat islets

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25-0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 +/- 1.4 to 4.0 +/- 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for less than 10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.     

Repeated isologous transplantation of islets of Langerhans in the diabetic rat.

The metabolic response to repeated isologous transplantation of isolated pancreatic islets was studied in 11 streptozotocin-diabetic AGUS rats. The islets were isolated with collagenase and transplanted intra-portally in batches previously found to be quantitatively insufficient for reversal of the diabetic state. Primary transplantation caused a slight improvement whereas retransplantation with the same number of islets three weeks later was followed by normalization of blood glucose, plasma insulin, urine volume and urine glucose per 24 hours during a three-month observation period. Repeated isologous transplantation of isolated pancreatic islets has an additive effect on the metabolism in that two insufficient tissue doses correct streptozotocin induced diabetes in the rats.     

Subcutaneous transplantation of islets into streptozocin-induced diabetic rats

Pancreatic islet transplantation into type 1 diabetic patients is currently being performed by intraportal infusion. This method, albeit reproducible, has some disadvantages including potential development of portal hypertension, hemorrhage, and an inability to retrieve or detect the transplanted tissue. Other transplant sites have been examined in animal models including the omentum, peritoneal cavity, and the spleen. A transplant site that has not been successful in supporting functional islet tissue transplantation in humans is the subcutaneous space due primarily to the lack of a well-defined vascular bed. This site has many favorable characteristics such as ease of access for transplantation and potential for removal of the transplanted tissue with a minimally invasive surgical procedure. This report addresses the evaluation of a subcutaneously placed device for the support of rat syngeneic islet transplantation in a streptozocin-induced diabetic model. The data generated support the use of this device for islet engraftment. In addition, beta cell function in this device compared favorably with the function of islets transplanted to the renal subcapsular space as well as islets within the native pancreas.     

Xenotransplantation of microencapsulated fetal rat islets

Fetal pancreatic islets were isolated from 21-day pregnant Wistar rats and enclosed in semipermeable alginate-polylysine-alginate capsules. Encapsulated islets that had been previously cultured for eight days in vitro were shown to secrete insulin in response to glucose challenge: low-glucose, high-glucose, and high-glucose + 3-isobutyl-1-methyl-xanthine (IBMX). Transplants of 800-1000 encapsulated cultured fetal islets into the peritoneal cavities of BALB/c mice with streptozotocin-induced diabetes restored normoglycemia for up to 171 days without immunosuppression. When the capsules were removed from 2 of the recipients they both quickly regressed to a diabetic state. Control groups of diabetic mice received unencapsulated, uncultured islets or empty capsules. The mortality rate among these animals was high and none experienced relief from hyperglycemia for longer than 6 days. These results demonstrate that cultured microencapsulated fetal rat islets of Langerhans can release insulin in response to an in vitro glucose challenge, and that transplants of these islets into diabetic mice can restore normoglycemia without the need for immunosuppressive therapy.     

微囊化猪胰岛肝动脉内移植治疗犬Ⅰ型糖尿病

目的　观察放射介入方法行微囊化猪胰岛细胞肝动脉肝内移植的可行性及有效性。方法　采用放射介入技术经股动脉穿刺将微囊猪胰岛经肝动脉植入糖尿病模型犬肝内 ,术后监测空腹血糖、C肽、胰岛素用量变化 ,行肝功能、肝脏组织病理学检查。结果　移植后糖尿病犬血清C肽水平升高 2～ 6倍 ,胰岛素用量逐步减少 ,3只犬停用胰岛素并维持空腹血糖正常 ;移植后肝转氨酶一过性升高 ,2周后降至正常。结论　经肝动脉肝内微囊猪胰岛素异种移植治疗移植Ⅰ型糖尿病犬安全、可行。     

Transplantation of isolated islets of Langerhans in diabetic dogs. II. The influence of histocompatibility.

By means of collagenase digestion and the Ficoll gradient separation technique, viable islets of Langerhans could be isolated from dog pancreata. The isolated islets were capable of secreting insulin after glucose stimulation. These islets of Langerhans were transplanted into the livers of diabetic dogs. We transplanted islets isolated from I donor pancreas for 1 kg body wt of the recipients. The dogs were made diabetic by subtotal pancreatectomy (80%) and two injections of 25 mg streptozotocin/kg body wt. In our experiments, we selected donor-recipient pairs by means of the mixed lymphocyte reaction macrophage electrophoretic migration inhibition (MLR-EM) test. In the group with a depression in the MLR-EM test of more than 15% (strong histoincompatibility), the mean survival time of the transplanted dogs was 46 ± 16 days. Another group of recipients with a depression of macrophage migration of less than 8% (weak histocompatibility) had a mean survival time of 135 ± 49 days after intraportal islet transplantation, and the blood glucose values could be normalized in this group for a period of 70 days. We found significant differences between both the groups in the glucose tolerance tests and in the IRI concentrations in the portal vein and in the vena cava superior. In the recipients, which received weakly histoincompatible islets, the IRI concentration in the superior vena cava was 64% of the normal value in the vena cava superior 4 weeks after intrahepatic islet transplantation. By means of the MLR-EM test, we were able to correlate inhibition with signs of rejection of the transplanted islets. Our experiments demonstrated that an allogeneic islet transplantation in diabetic dogs across a weakly histocompatible barrier can improve the results in comparison to the islet transplantation across a strongly histocompatible barrier. However, this typing maneuver alone is not able to prevent the islet rejection.     

Reversal of diabetes in dogs by transplantation of pure cryopreserved islets

We have autotransplanted highly purified cryopreserved canine islets into pancreatectomized dogs. Islets were frozen by slow cooling (0.25 degrees C/min) to -40 degrees C, stored at -196 degrees C and thawed rapidly (200 degrees C/min). A total of 7868 +/- 665 (means +/- SE) cryopreserved islets/kg body weight were implanted to the spleen (n = 7) by venous reflux, and seven other control dogs received 6811 +/- 653 fresh islets/kg (P versus cryo., NS). Fasting plasma glucose [( PG] mg/dl, +/- SEM) and intravenous glucose tolerance were determined before and at 1 and 3 months postimplantation. Six dogs that received cryopreserved islets and all seven control animals promptly became normoglycemic, with PGs of 133 +/- 23 and 110 +/- 4, respectively, at 1 week postimplantation (NS). During follow-up one normoglycemic animal from each group died due to small bowel volvulus and one recipient of fresh islets became diabetic at 14 days. The remaining dogs remained normoglycemic with PGs of 89 +/- 3 and 92 +/- 3 in dogs receiving cryopreserved and fresh islets, respectively, at 3 months postimplantation (NS). Mean K value (decline of glucose, %/min +/- SEM) at ivGTT for all dogs was 2.5 +/- 0.3 preoperatively. At 1 and 3 months postimplantation, the K values of dogs receiving cryopreserved islets were 1.3 +/- 0.1 and 1.4 +/- 0.2, respectively, compared with 1.3 +/- 0.2 and 2.0 +/- 0.2 for control dogs (NS). These data demonstrate prompt and sustained function of a defined quantity of frozen-thawed purified islets in a large mammal. Cryopreservation is an effective method for the long-term storage of purified islet tissue.     

微囊化大鼠胰岛异种移植的实验研究

目的　通过动物实验明确微囊化胰岛是否具有免疫隔离作用。方法　SD大鼠胰腺原位消化 ,Ficoll间断密度梯度离心法纯化、分离胰岛 ,气流吹喷制作海藻酸钠 /聚赖氨酸 /海藻酸钠(APA)微囊化大鼠胰岛 ,比较微囊化与未微囊化胰岛的胰岛素释放试验 ;将微囊化 (实验组 )与未微囊化 (对照组 )大鼠胰岛植入链脲佐菌素 (STZ)诱导的I型糖尿病小鼠中 ,作两组间血糖正常持续时间比较。结果　实验组与对照组的胰岛素释放试验差异无显著性 (P >0 .0 5 ) ;实验组血糖正常持续时间为 2 3～ 6 5d(平均 48d) ,对照组为 3～ 6d(平均 5d) ,两组差异有极显著性 (P <0 .0 1)。已排斥的实验组小鼠腹腔灌洗发现部分微囊化胰岛存活 ,部分已坏死 ,但微囊膜皆完整 ,囊壁无纤维化。结论　微囊具有良好的免疫隔离作用 ,可使胰岛移植物存活时间明显延长。同时推测微囊内移植物死亡与细胞因子、自由基作用或营养不足等有关。     

改良的微囊化新生猪胰岛细胞移植治疗Ⅰ型糖尿病八例报告

在动物实验的基础上，对８例Ⅰ型糖尿病患者进行了改良的微囊化新生猪胰岛细胞移植治疗。８例患者中，３例移植于网膜囊，５例移植于三角肌，于移植前及移植后１、６、１２个月观察空腹血糖（ＦＢＧ）、糖基化血红蛋白Ａ１ｃ（ＨｂＡ１ｃ）、Ｃ－酞、ＣＤ４阳性细胞、ＣＤ８阳性细胞、可溶性白细胞介素２受体和胰岛素用量（ＩＮＳ）的变化。结果显示，移植前后Ｔ细胞亚群无明显变化，提示无排斥反应发生；结果还显示移植的猪胰岛数量决定了疗效：移植量少于１０个猪胰的胰岛（约３２６１８０±１２１３０个胰岛），移植后ＩＮＳ减量少于４０％，Ｃ－酞升高不明显，移植量达到１０个猪胰的胰岛，术后ＩＮＳ减量＞７５％，Ｃ－酞峰值是术前的２．５６倍。提示此种方法移植的必需量为１０个新生猪胰的胰岛。     

微囊新生猪胰岛细胞体外培养的研究

用胶原酶消化和体外培养的方法成功地获得了大量有活性和较纯的新生猪胰岛细胞，用微囊包膜技术包裹胰岛细胞，并进行培养。由放射免疫药盒对培养液中胰岛素的含量测定，胰岛素释放试验和组织学检查等体外实验所得的数据，证实了微囊和非微囊的新生猪胰岛的活性和分泌功能的差异不显著，说明本包膜材料和一步法制囊新技术对胰岛细胞无损伤，微囊包膜后胰岛细胞的活性和分泌功能良好。     

微囊化猪胰岛的免疫原性研究

目的 经过体外实验,明确微囊化新生猪胰岛样细胞团（ＩＣＣ）的免疫原性。方法 通过胰岛－淋巴细胞混合培养（ＭＬＩＣ）后的淋巴细胞转化试验。以游离的新生猪ＩＣＣ和空微囊为对照,比较微囊ＩＣＣ组与对照组的淋巴细胞转化值。结果 微囊ＩＣＣ组淋巴细胞转化值显著低于游离ＩＣＣ组（Ｐ〈０．０５）,但明显高于空囊组（Ｐ〈０．０５）。结论 微囊包裹后的新生猪ＩＣＣ的免疫原性比游离的新生猪ＩＣＣ的免疫原性显著降低。提     

Cytokine-induced functional suppression of microencapsulated rat pancreatic islets in vitro

BACKGROUND: It is likely that inflammatory cytokines are released near microencapsulated islets in vivo. METHODS: Rates of insulin release or glucose oxidation were measured after culture of microencapsulated rat islets with interleukin (IL)-1beta and tumor necrosis factor-(TNF-alpha). Their ability to recover from IL-1beta-induced suppression was also investigated. RESULTS: Microencapsulated islets were suppressed after exposure to IL-1beta, which was potentiated by TNF-alpha. After exposure to lower IL-1beta concentrations, microencapsulated islets had similar oxidation rates as corresponding controls. At higher concentrations, microencapsulated islets were more suppressed than nonencapsulated islets. Microencapsulated and control islets were able to recover from suppression after exposure to 2.5 U/ml of IL-1beta. CONCLUSIONS: Microencapsulation using the present alginate/poly-L-lysine/alginate capsules does not protect islets against the detrimental effects of IL-1beta and TNF-alpha. Indeed, microencapsulated islets seem to be more susceptible to suppression at higher concentrations of IL-1beta. However, after exposure to a lower concentration of IL-1beta, microencapsulated islets can recover.     

胎肝胰岛细胞联合移植治疗糖尿病大鼠

目的探讨胎肝胰岛细胞联合移植治疗糖尿病大鼠的效果,以及胎肝细胞移植诱导糖尿病受者鼠免疫耐受的可行性.方法建立SD大鼠糖尿病模型,将实验动物随机分为正常对照组、糖尿病对照组、胰岛细胞组及肝细胞胰岛细胞联合移植组.将SD胎鼠的胰岛细胞移植于受者浆膜下,观察受者鼠的糖尿病病情变化及免疫状态.结果移植后58.8%(20/34)动物糖尿病得到缓解.联合移植组移植胰岛细胞存活期较胰岛细胞组明显延长(P<0.01);移植后1周,联合移植组血清胰岛细胞肽水平明显高于胰岛细胞组和糖尿病对照组(P＜0.01);脾细胞产生白细胞介素2(IL-2)的活性联合移植组显著低于胰岛细胞组(P＜0.001).结论胃浆膜下是大鼠胰岛移植较理想的移植部位;肝细胞移植可以明显延长移植胰岛细胞的功能存活期,并诱导受者产生特异性免疫耐受和非特异性免疫抑制.     

微囊化人胎胰岛功能的体外观察

目的 :观察微囊的通透性及对微囊化胰岛分泌功能的影响。方法 :将用胶原酶消化获得的胰岛细胞分为 2份 ,1份微囊包膜 ,1份未予包膜作为对照 ,培养 7天 ,比较两组培养液中胰岛素含量并分别用2 .7mmol·L-1、16 .6mmol·L-1浓度的葡萄糖及 16 .6mmol·L-1的葡萄糖加 10mmol·L-1茶碱刺激胰岛B细胞 ,观察其胰岛素分泌功能。结果 :两组在培养液中胰岛素含量及葡萄糖、茶碱刺激的胰岛B细胞胰岛素分泌方面无显著性差异 (P >0 .0 5 )。结论 :该微囊具有良好的通透性 ,微囊化胰岛的活性和分泌功能未受成囊过程的影响