Influence of VEGF on the viability of encapsulated pancreatic rat islets after transplantation in diabetic mice

After pancreatic islet transplantation, insufficient blood supply is responsible for the loss of islet viability. The aim of our study was: 1) to determine the influence of vascular endothelial growth factor (VEGF) on the survival of encapsulated rat islets transplanted into healthy and diabetic mice and 2) to evaluate the metabolic efficiency of the VEGF-supplemented grafts. Twenty-four hours after culture, 50 rat islets immobilized into collagen in the presence of VEGF (100 ng/ml) and encapsulated (AN69 membrane, HOSPAL) were grafted in the peritoneal cavity of healthy or streptozotocin-induced diabetic mice (n = 6). Seven, 14, and 28 days after implantation, the encapsulation device and tissue surrounding the device were removed and the following parameters were analyzed: the number and the diameter of buds, the distance between devices and buds, the amount of cellular adhesion on the capsule surface, and the level of insulin secreted by encapsulated islet. For reversal of diabetes, 1000 rat islets encapsulated in the presence of VEGF were implanted in the peritoneal cavity of diabetic mice and fasting glycemia was analyzed. After 7 days of islet implantation in the absence of VEGF, the bud diameter was 16.1 +/- 6.9 microm in diabetic mice and 34.4 +/- 3.9 microm in healthy mice. However, the number of buds increased by a factor 2.5 in the presence of VEGF in both types of mice. Furthermore, when islets were transplanted in the presence of VEGF, the distance between the device and the buds was significantly decreased in both types of mice (p < 0.001) after 7, 14, and 28 days of islet implantation. Capsule analysis showed a decrease in cellular adhesion when the islets were encapsulated in the presence of VEGF. Insulin secretion of the islets was higher in the presence of VEGF compared with islets alone at all steps of the study. When 1000 rat islets were transplanted in the presence of VEGF, the glycemia level decreased to 6.2 +/- 0.8 mmol/L after 3 days and remained stable until at least 28 days. In contrast, in the absence of VEGF, the initial decrease in the glucose level was rapidly followed by a relapse in hyperglycemia. In summary, VEGF increased the viability of engrafted encapsulated islets, increasing the duration of a normalized glycemia in diabetic mice following transplantation. Local adjunction of VEGF may therefore improve the clinical outcome of islet transplantation.     

Long-term graft function after allogeneic islet transplantation

Islet transplants are emerging as a viable option for the treatment of type 1 diabetes mellitus. From 1989 to 1995 we conducted a series of simultaneous islet-kidney transplants in six uremic type 1 diabetic patients. We report two of these patients who have shown persistent islet graft function over many years. Two female patients with duration of diabetes of 27 and 37 years underwent simultaneous islet-kidney transplant under steroid- and cyclosporine-based immunosuppression. Freshly isolated islets were supplemented with cryopreserved islets from our low-temperature bank of frozen islets. A total islet mass of 9,866 and 15,061 islet equivalents/kg body weight, respectively, was transplanted into the liver through portal vein. Reasonable blood glucose control has been achieved for up to 6 years posttransplant in one patient, but there was minimum clinical benefit from the islet graft at 10 years. In contrast, sustained insulin secretion with nearly normal HbA1c at 13 years follow-up was observed in another patient, providing hope for improving long-term graft outcomes for islet transplant recipient.     

骨髓来源细胞移植对糖尿病小鼠胰岛功能的影响

目的 观察骨髓来源细胞(BMDCs)移植对糖尿病小鼠胰岛功能的影响.方法 建立糖尿病小鼠模型并分成两组:实验组小鼠(n=8)通过尾静脉移植BMDCs;对照组小鼠(n=8)通过尾静脉注射磷酸盐缓冲液(PBS).观察两组小鼠血糖的变化、胰岛数量、胰腺组织形态学特征及相关标记物的表达.结果 与对照组比较,实验组小鼠移植后第4周血糖出现明显下降(20.7±5.2)比(27.1±1.4)mmol/L,P＜0.05,并持续下降至第6周(16.9±6.0)比(27.7±0.3)mmol/L,P＜0.01,胰岛数目显著增加(22.9±4.8)比(11.6±5.2)个,P＜0.01;实验组小鼠胰岛周围和胰岛内发现绿色荧光蛋白(GFP)阳性细胞,部分GFP阳性细胞同时表达CD34,但未发现同时表达GFP和insulin的细胞.结论 BMDCs移植能促进糖尿病小鼠胰岛的修复和再生,但BMDCs在糖尿病小鼠体内不能转分化为胰岛β细胞,CD34阳性细胞在损伤胰岛修复和再生的过程中起重要作用.     

No transmission of porcine endogenous retrovirus after transplantation of adult porcine islets into diabetic nude mice and immunosuppressed rats

BACKGROUND: The aim of this study was to investigate whether transmission of porcine endogenous retrovirus (PERV) occurs in a model of diabetes reversal by the xenotransplantation of adult porcine islets (APIs) into immunoincompetent diabetic rodents. METHODS: Black-6 nu/nu mice and Lewis rats were immunosuppressed with cyclosporin A (CsA) and FTY 720, and rendered diabetic with streptozotocin. Purified APIs were transplanted into the renal subcapsular space; 5,000 islet equivalents (IEQs) were used in the nude mice (n = 4) and 40,000 IEQs in the rats (n = 4). The nude mice were sacrificed at 75 days after transplantation. In order to confirm chronic xenograft function, the graft-bearing kidney was removed prior to sacrifice. The rats were followed until xenograft rejection, at which time they were sacrificed. Immediately after sacrifice, tissue samples (liver, spleen, and small intestine) were taken for analysis. Quantitative polymerase chain reaction (PCR) was used to assess evidence of PERV transmission, and porcine cell chimerism. RESULTS: All animals became normoglycemic within 48 h of transplantation. The nude mice remained normoglycemic during the 75-day study period, with removal of the graft-bearing kidney resulting in prompt hyperglycemia. The rats remained normoglycemic until xenograft rejection, which occurred at 66 +/- 28 days. Despite the evidence of porcine cell microchimerism in recipients, real-time PCR detected no evidence of PERV transmission in any of the tissue specimens tested. CONCLUSIONS: There was no evidence of PERV transmission following transplantation of pig islets into diabetic nude mice and immunosuppressed rats.     

Intraportal transplantation of allogenic pancreatic islets encapsulated in barium alginate beads in diabetic rats

The survival of microencapsulated islets transplanted into the unmodified peritoneal cavity is limited, even if capsular overgrowth is restricted to a minimum, due to an insufficient oxygen supply to the islets. Therefore, research efforts should focus on finding or creating a transplantation site, which permits a closer contact between the encapsulated islets and the blood. For this reason, the liver could be an interesting candidate. The aim of the present study was to test the hypothesis that the intraportal transplantation of allogenic islets encapsulated in small-sized barium alginate beads is safe and succeeds to induce normoglycemia in diabetic rats. The intraportal transplantation of 1,500 islets encapsulated in barium alginate beads leads within 10 h and up to 24 h to blood sugar concentrations below 40 mg/dL, most likely due to an acute cell lysis of the graft. Afterwards, the reappearance of the diabetic state could be detected in these animals. Most likely these findings are induced by a sudden hypoxia to the islets. We believe that the occlusion of small- and medium-sized portal venules by the alginate beads is responsible for this effect. Therefore, in forthcoming studies, barium alginate beads, with a diameter below 350 micro m, stabilized with medical approved additives should be used.     

Long-term graft and patient survival following renal transplantation in diabetic patients

OBJECTIVE: To study long-term graft and patient survival following renal transplantation in diabetic and non-diabetic patients. MATERIAL AND METHODS: Over the time period 1985-99, 498 transplantations in 399 non-diabetic patients and 68 transplantations in 62 diabetic patients were performed. The groups were similar with respect to age and sex. RESULTS: The patient survival rates (diabetic versus non-diabetic patients) were 88% vs 91% (p=NS) at 1 year, 68% vs 73% (p=NS) at 5 years and 31% vs 52% (p<0.05) at 10 years. The graft survival rates (diabetic versus non-diabetic patients) were 72% vs 72% at 1 year, 52% vs 52% at 5 years and 27% vs 33% (p=NS) at 10 years. In the diabetic patients, mean haemoglobin (Hb)A1c 2 years before and 2 years after the transplantation was 7.5+/-1.4 vs 8.2+/-1.6 mmol/l (p<0.05) and the mean blood pressure was 112+/-12 vs 107+/-9 mmHg (p<0.05). Of the diabetic patients, 55% were smokers. Among the diabetic patients, graft and patient survival were independent of smoking habits, blood pressure, HbA1c and total cholesterol. CONCLUSIONS: Graft survival was similar in diabetic and non-diabetic patients. For the first 5 years following renal transplantation, the patient survival rates in the two groups were similar. Thereafter, survival among diabetic patients was poor. Mean HbA1c was relatively high, especially after the transplantation, and this may have contributed to the more rapid progression of cardiovascular disease seen in diabetic patients with nephropathy.     

Experimental transplantation with principal islets of teleost fish (Brockmann bodies). Long-term function of tilapia islet tissue in diabetic nude mice.

Certain teleost fish have macroscopically visible islets called BBs that are anatomically discrete. BBs were harvested from Oreochromis nilotica (tilapia) with microscissors, divided, and cultured overnight at 37 degrees C before transplantation into STZ-induced diabetic nude mice. Each mouse received BB fragments from 3-5 fish weighing in aggregate approximately 1.7 kg. Non-FPGs were monitored 5 days/wk. Recipients remained normoglycemic (plasma glucose < 11.1 mM) for 50 days posttransplantation. Mice bearing 50-day-old grafts had essentially normal GTTs. Left nephrectomies then were performed to remove the grafts, and plasma glucose levels in recipient mice rose to > 22.2 mM. Histological examination of graft-bearing kidneys showed viable, vascularized islet tissue containing numerous well-granulated beta-cells; examination of recipient native pancreases revealed small islets composed predominantely of non-beta-cells.     

Long-term cryogenic storage of purified adult human islets of Langerhans

Reliable high-recovery human islet storage would facilitate tissue matching, organ sharing, and immune manipulation of donor islets and prospective diabetic recipients. Collagenase-isolated, Ficoll-purified pancreatic islets (median 21,000, 15% of total islet yield) from eight cadaver pancreases were cultured in vitro for 24 h, equilibrated in three steps with dimethyl sulfoxide (DMSO) to a 2-M concentration, supercooled, nucleated, and cooled at 0.25 degree C/min to -40 degrees C before storage at -196 degrees C for 44.25 +/- 8.75 days. Rewarming at 200 degrees C/min and removal of DMSO with 0.75 M sucrose preceded 48 h of culture and retesting. Recovery postthaw by microscope count on duplicate aliquots was 94.2 +/- 3.5% of prefreeze counts and by triplicate assay of extractable insulin was 90.0 +/- 22.3% on day 0 and 74.1 +/- 12.6% after a 48-h culture. Nonfrozen islets increased basal insulin secretion 7.7 +/- 2.8 times after stimulation with 300 mg/dl glucose in perifusion, whereas islets frozen-thawed and cultured 48 h increased 6.2 +/- 0.8 times (NS). Peak stimulated insulin release was 0.92 +/- 0.14 microU.islet-1.min-1 before storage and 0.73 +/- 0.14 microU.islet-1.min-1 (79% of control, NS) after freeze-thaw and a 48-h culture. Total insulin secretion (area under curve) was 66% of prefreeze values at 48 h. Immunocytochemical stains revealed preservation of islet morphology postthaw. Electron microscopy showed intact cellular and nuclear membranes and intracellular organelles. Frozen-thawed islets harvested 14 days after renal subcapsular xenografting in nude mice were revascularized and well granulated. Cryopreservation can achieve prolonged storage of large numbers of human islets with high recovery numerically and functionally, making this a feasible approach for future trials of human islet transplantation.     

Prolonged glucose normalization of streptozotocin-induced diabetic mice by transplantation of rat islets coencapsulated with crosslinked hemoglobin

BACKGROUND: Facilitated oxygen transport by crosslinked hemoglobin (Hb-C) in islet microcapsules may promote transplanted graft function by improving islet functionality and viability. METHODS: This study investigated the in vivo efficacy of Hb-C as an oxygen carrier on the functionality and viability of microencapsulated rat islets. Hb-C by poly(ethylene glycol) was introduced into rat islet microcapsules (alginate-poly[L-lysine]-alginate microcapsule), and 500 suboptimal encapsulated islets were xenotransplanted into each streptozotocin-induced diabetic BALB/c mouse. The graft efficacy over time was evaluated by measuring nonfasting blood glucose level, body weight, and glucose tolerance. RESULTS: Mice that received Hb-C-containing microcapsules maintained normoglycemia for at least 8 weeks with normal glucose clearance, determined by intraperitoneal glucose tolerance test. However, the mice that received the conventional control islet microcapsule (without Hb-C) transplant showed graft failure in 4 weeks, exhibited by hyperglycemia, weight loss, and deteriorated glucose tolerance. Severe central necrosis of retrieved islets was observed for the control islet capsule graft after 8 weeks. CONCLUSION: The present study revealed that the incorporation of Hb-C in islet microcapsules promotes graft function for a longer period of time than the conventional islet capsules. Therefore, Hb-C coencapsulation is a potential approach for prolonging graft function of islet microcapsules and reducing the number of islets required for normoglycemia.     

糖尿病小鼠胰岛移植部位的实验研究

目的探讨小鼠肝内、肾包膜下等不同部位胰岛移植物生存时间及免疫学功能的差异。方法采用滤网法分离纯化胰岛,供体胰岛植入受体肝内及肾包膜下。用酶联免疫吸附试验 (ELISA)测定INF-γ的含量。3H-胸腺嘧啶脱氧核苷(3H-TdR)掺入法测定单向混合淋巴细胞反应。结果肾包膜下移植组胰岛生存时间(12．18±1．25)d明显长于肝内移植组[(7．09±0．70)d,P< 0．01]。单项混合淋巴细胞反应显示,肝内移植组淋巴细胞较肾包膜下移植组增殖明显(P<0．01)。上清液测IFN-γ含量,肝内移植组明显升高,差别有统计学意义(P<0．05)。结论不同移植部位, 胰岛的生存时间及免疫功能差异存在统计学意义,肾包膜下是胰岛移植的理想部位。     

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     

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.     

微囊化大鼠肝细胞移植的组织学研究

目的 研究微囊化大鼠肝细胞腹腔移植后的存活情况以及组织学改变。方法 用二步法胶原酶门－腔静脉灌注法分离Wistar大鼠肝细胞,用Percoll梯度分离液纯化,海酸钠－氯化钡法微囊化包裹肝细胞,分别行腹腔注射移植,将纯化的肝细胞移植至SD大鼠体内（第1组）、微囊化包裹的肝细胞移植至SD大鼠体内（第2组）及Wistar大鼠体内（第3组）,观察移植后各组不同时间肝细胞存活率及其组织学变化。结果 （1）移植后第4、7d,各组间肝细胞存活率的差异均有显著性（P＜0.01）;移植后第14d,第2组与经3组间肝细胞存活率的差异无显著性;（2）移植后第4d开始,微囊周围出现纤维增生现象,第2组较第3组明显。结论 微囊化可为移植的肝细胞提供免疫屏蔽作用,从而提高肝细胞移植的存活率;微囊周围纤维增生可影响肝细胞的存活率。     

Long-term survival after kidney and kidney-pancreas transplantation in diabetic patients

PURPOSE: To investigate the influence of diabetes mellitus on patient and graft survival among renal versus renal-pancreatic recipients. METHODS: Among 270 renal transplants performed from 1985 to 2002, a total of 204 (75%) were in diabetic patients and 66 (25%) in nondiabetic patients. Among the 204 diabetic patients 161 (60%) kidneys were transplanted simultaneously with a pancreatic graft (SKPT group). The overall group of patient included 164 (61%) men and 106 (39%) women with mean time on dialysis of 31 +/- 21 months (range 0 to 126 months). The mean duration of diabetes was 24 +/- 7 years (range 5 to 51 years). Ninety-nine percent of the patients were on renal replacement therapy (79% hemodialysis and 20% peritoneal dialysis). RESULTS: The overall rejection rate was similar (NS). Both patient and kidney graft survival rates were worse in diabetics. Patient survival was 82% at 5 years among patients undergoing SKPT, 60% in diabetics receiving only a kidney, and 88% in nondiabetic transplanted patients. Kidney graft survival at 5 years was 77% in diabetics receiving SKPT, 68% in diabetics receiving a kidney alone, and 82% in nondiabetic patients. Overall patient survival was significantly greater among nondiabetics (P =.002) or in diabetics who received SKPT compared with diabetics who only had a kidney transplant (P =.001). CONCLUSIONS: This retrospective clinical evaluation confirms that combined pancreas and kidney transplantation should be the first choice to insulin-dependent diabetes mellitus (IDDM) patients with end-stage diabetic nephropathy.