Prevention of core cell damage in isolated islets of Langerhans by low temperature preconditioning

AIM: To study the core cell damage in isolated islets of Langerhans and its prevention by low temperature preconditioning (26 ℃).METHODS: Islets were cultured at 37 ℃ for 7-14 d after isolation, and then at 26 ℃ for 2, 4 and 7 d before additional culture at 37 ℃ for another 7 d. Core cell damage in the isolated islets was monitored by video-microscopy and analyzed quantitatively by use of a computer-assisted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated islets and the area of the core cell damage that developed in those islets over time during culture. Histology and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize the cell damage and to monitor islet function.RESULTS: Microscopic analysis showed that during the 7 to 14 d of culture at 37 ℃, core cell damage occurred in the larger islets with diameters ＞200 μm, which included both necrotic and apoptotic cell death. Low temperature (26 ℃) culture could prevent core cell damage of isolated islets. The 7-d culture procedure at 26 ℃ could inhibit most of the core cell (excluding diameters＞300 μm) damages when the islets were re-warmed at 37 ℃.CONCLUSION: Our results indicate that core cell damage within isolated islets of Langerhans correlates with the size of islets. Low temperature (26 ℃) culture can prevent core cell damage in isolated islets, and successfully precondition these islets for incubation at 37 ℃. These novel findings may help to understand the pathophysiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.

Survival of encapsulated islets: More than a membrane story

At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets ofLangerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane(e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, especially when using large retrievable macrocapsules implanted in poorly oxygenated sites like the subcutis. Notably, islets can survive under low oxygen tension and are physiologically active at > 40 Torr. Surprisingly, when densely crowded, islets are fully functional under hyperoxic pressure of up to 500 Torr(> 300% of atmospheric oxygen tension). The review also addresses an additional category of requirements for optimal performance of transplanted islets, named auxiliary technologies. These include control of inflammation, apoptosis, angiogenesis, and the intra-capsular environment. The review highlights that curing diabetes with a functional bio-artificial pancreas requires optimizing all of these aspects, and that significant advances have already been made in many of them.

Adult islets cultured in collagen gel transdifferentiate into duct-like cells

AIM: To establish a model of islet-ductal cell transdifferen-tiation to identify the transdifferentiated cells. METHODS: Collagen was extracted from rat tail at first. Purified rat islets were divided into three groups, embedded in collagen gel and incubated respectively in DMEM/F12 alone (control group), DMEM/F12 plus epidermal growth factor (EGF), DMEM/F12 plus EGF and cholera toxin (CT). Transdifferentiation was proved by microscopy, RT-PCR, immunohistochemistry and RIA. RESULTS: Islets embedded in collagen gel plus EGF and CT were cystically transformed and could express new gene cytokeratin 19 while still maintaining the expression of insulin and Pdx-1 genes. Immunohistochemistry demonstrated that the protein of cytokeratin 19 was only expressed in the third group. The insulin content secreted by islets in the third group decreased significantly during the transdiffe-rentiation. CONCLUSION: CT is a crucial factor for the islet-ductal cell transdifferentiation.

Intracerebral xenotransplantation of semipermeable membrane- encapsuled pancreatic islets

AIM： To identify the decreasing effect of xenotransplantion in combination with privileged sites on rejection and death of biological semipermeable membrane-（BSM） encapsulated implanted islets. METHODS： After the BSM experiment in vitro, BSM- encapsulated SD rat＇s islet-like cell clusters （ICCs） were xenotransplanted into normal dog＇s brain. Morphological changes were observed under light and transmission electron microscope. The islets and apoptosis of implanted B cells were identified by insulin-TONEL double staining. RESULTS： The BSM used in our study had a favorable permeability, some degree of rigidity, lighter foreign body reaction and toxicity. The grafts consisted of epithelioid cells and loose connective tissue. Severe infiltration of inflammatory cells was not observed. The implanted ICCs were identified 2 mo later and showed typical apoptosis. CONCLUSION： BSM xenotransplantation in combination with the privileged site can inhibit the rejection of implanted heterogeneous ICCs, and death of implanted heterogeneous B cells is associated with apoptosis. 2005 The WJG Press and Elsevier Inc. All rights reserved.

Regenerative medicine of pancreatic islets

The pancreas became one of the first objects of regenerative medicine,since other possibilities of dealing with the pancreatic endocrine insufficiency were clearly exhausted.The number of people living with diabetes mellitus is currently approaching half a billion,hence the crucial relevance of new methods to stimulate regeneration of the insulin-secretingβ-cells of the islets of Langerhans.Natural restrictions on the islet regeneration are very tight;nevertheless,the islets are capable of physiological regeneration viaβ-cell self-replication,direct differentiation of multipotent progenitor cells and spontaneousα-toβ-orδ-toβ-cell conversion(trans-differentiation).The existing preclinical models ofβ-cell dysfunction or ablation(induced surgically,chemically or genetically)have significantly expanded our understanding of reparative regeneration of the islets and possible ways of its stimulation.The ultimate goal,sufficient level of functional activity ofβ-cells or their substitutes can be achieved by two prospective broad strategies:β-cell replacement andβ-cell regeneration.The“regeneration”strategy aims to maintain a preserved population ofβ-cells through in situ exposure to biologically active substances that improveβ-cell survival,replication and insulin secretion,or to evoke the intrinsic adaptive mechanisms triggering the spontaneous non-β-toβ-cell conversion.The“replacement”strategy implies transplantation ofβ-cells(as non-disintegrated pancreatic material or isolated donor islets)orβ-like cells obtained ex vivo from progenitors or mature somatic cells(for example,hepatocytes orα-cells)under the action of small-molecule inducers or by genetic modification.We believe that the huge volume of experimental and clinical studies will finally allow a safe and effective solution to a seemingly simple goal-restoration of the functionally activeβ-cells,the innermost hope of millions of people globally.

Macro-or microencapsulation of pig islets to cure type 1 diabetes

Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply;several human pancreas donors are often needed to treat one diabetic recipient;the intrahepatic site may not be the most appropriate site for islet implantation;and immunosuppressive regimens,which are associated with side effects,are often required to prolong survival of the islet graft.An alternative source of insulinproducing cells would therefore be of major interest.Pigs represent a possible alternative source of beta cells.Grafting of pig islets may appear difficult because of the immunologic species barrier,but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression.Therefore,a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation.Although several groups have shown that encapsulated pig islets are functional in small-animal models,less is known about the use of bioartificial pancreases in large-animal models.In this review,we summarize current knowledge of encapsulated pig islets,to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.

Glucose-stimulated insulin secretion in isolated pancreatic islets: Multiphysics FEM model calculations compared to results of perifusion experiments with human islets

Because insulin released by the β-cells of pancreatic islets is the main regulator of glucose levels, the quantitative modeling of their glucose-stimulated insulin secretion is of obvious interest not only to improve our understanding of the processes involved, but also to allow better assessment of β -cell function in diabetic patients or islet transplant recipients as well as the development of improved artificial or bioartificial pancreas devices. We have recently developed a general, local concentrations-based multiphysics computational model of insulin secretion in avascular pancreatic islets that can be used to calculate insulin secretion for arbitrary geometries of cultured, perifused, transplanted, or encapsulated islets in response to various glucose profiles. Here, experimental results obtained from two different dynamic glucose-stimulated insulin release (GSIR) perifusion studies performed by us following standard procedures are compared to those calculated by the model. Such perifusion studies allow the quantitative assessment of insulin release kinetics under fully controllable experimental conditions of varying external concentrations of glucose, oxygen, or other compounds of interest, and can provide an informative assessment of islet quality and function. The time-profile of the insulin secretion calculated by the model was in good agree- ment with the experimental results obtained with isolated human islets. Detailed spatial distributions of glucose, oxygen, and insulin were calculated and are presented to provide a quantitative visualization of various important aspects of the insulin secretion dynamics in perifused islets.

On Damped Wave Diffusion of Oxygen in Pancreatic Islets: Parabolic and Hyperbolic Models

Damped wave diffusion effects during oxygen transport in islets of Langerhans is studied. Simultaneous reaction and diffusion models were developed. The asymptotic limits of first and zeroth order in Michaelis and Menten kinetics was used in the study. Parabolic Fick diffusion and hyperbolic damped wave diffusion were studied separately. Method of relativistic transformation was used in order to obtain the solution for the hyperbolic model. Model solutions was used to obtain mass inertial times. Convective boundary condition was used. Sharma number (mass) may be used in evaluating the importance of the damped wave diffusion process in relation to other processes such as convection, Fick steady diffusion in the given application. Four regimes can be identified in the solution of hyperbolic damped wave diffusion model. These are;1) Zero Transfer Inertial Regime, 0 0≤τ≤τinertia;2) Rising Regime during times greater than inertial regime and less than at the wave front, Xp > τ, 3) at Wave front , τ = Xp;4) Falling Regime in open Interval, of times greater than at the wave front, τ > Xp. Method of superposition of steady state concentration and transient concentration used in both solutions of parabolic and hyperbolic models. Expression for steady state concentration developed. Closed form analytic model solutions developed in asymptotic limits of Michaelis and Menten kinetic at zeroth order and first order. Expression for Penetration Length Derived-Hypoxia Explained. Expression for Inertial Lag Time Derived. Solution was obtained by the method of separation of variables for transient for parabolic model and by the method of relativistic transformation for hyperbolic models. The concentration profile was expressed as a sum of steadty state and transient parts.

Islet cell transplantation as a cure for insulin dependent diabetes: current improvements in preserving islet cell mass and function

OBJECTIVE: To review the current progress of islet cell transplantation in patients with insulin-dependent diabetes, emphasizing on the difficulties with recovering and preserving islet cell mass and function, 30% of which is lost during the peri-transplantation period. RESULTS: The islet-cell isolation technique is perfected, but improvements are still progressing in two major directions: preservation of islet cells and tolerance induction. Optimum islet cell viability and function depends on appropriate revascularization of the islet graft and blockade of thrombus formation as well as cytokine and free radical release. Conditioning the islet cells in-vitro prior to transplantation to either upregulate VEGF expression or downregulate NF-kappa B transcription factor has proven to improve revascularization and to prevent islet cell apoptosis and cytokine-mediated damage. Tolerance induction is currently being best achieved by selecting and combining immunosuppressive agents such as monoclonal antibodies which target the major signaling molecules during immune activation, but which are least toxic to islet cells. CONCLUSIONS: Patients with insulin-dependent diabetes will greatly benefit from current developments in effective approaches to protect islets during the peritransplant period. Emerging interest in stem cell biology and differentiation may provide the ultimate solution to the problem of organ scarcity and islet cell protection from the peritransplant induced damage.

γ-aminobutyric acid secreted from isletβ-cells modulates exocrine secretion in rat pancreas

AIM： To investigate the role of endogenous γ-aminobutyric acid （GABA） in pancreatic exocrine secretion. METHODS： The isolated, vascularly perfused rat pancreas was employed in this study to eliminate the possible influences of extrinsic nerves and hormones. Cholecystokinin （CCK; 10 pmol/L） was intra-arterially given to stimulate exocrine secretion of the pancreas. RESULTS： Glutamine, a major precursor of GABA, which was given intra-arterially at concentrations of 1, 4 and 10 mmol/L, dose-dependently elevated the CCK-stimulated secretions of fluid and amylase in the normal pancreas. Bicuculline （10 μmol/L）, a GABAA receptor antagonist, blocked the enhancing effect of glutamine （4 mmol/L） on the CCK-stimulated exocrine secretions. Glutamine, at concentrations of 1, 4 and 10 mmol/L, dose-dependently increased the GABA concentration in portal effluent of the normal pancreas. The effects of glutamine on the CCK-stimulated exocrine secretion as well as the GABA secretion were markedly reduced in the streptozotocintreated pancreas. CONCLUSION： GABA could be secreted from β-cells into the isletoacinar portal system after administration of glutainine, and could enhance the CCK-stimulated exocrine secretion through GABAA receptors. Thus, GABA in islet β-cells is a hormone modulating pancreatic exocrine secretion.

Protective effect of glucocorticoid-free immunosuppressive regimen in allogenic islet transplantation

BACKGROUND: The most common complication after allogenic islet transplantation is rejection. This study was to evaluate the effect of anti-rejection of glucocorticoid-free immunosuppressive regimen on allogenic islet transplantation. METHODS: Tacrolimus(FK506)+mycophenolate mofetil (MMF) and FK506+MMF+prednisone (Pred) were administered respectively for 2 weeks to inhibit rejection after allogenic islet transplantation in rats, which were compared with the control group. The concentrations of blood glucose, insulin and C-peptide were determined dynamically in recipients and the sites of transplantation were observed morphologically. RESULTS: As compared with the control group without immunosuppressive agents, FK506+MMF and FK506+MMF+Pred could prolong the survival time of grafts significantly. There were many morphologically intact islets in the liver of recipients 2 months after transplantation. Group FK506+MMF kept normal levels of blood glucose, insulin and C-peptide beyond 60 days after transplantation. In contrast, group FK506+MMF+Pred secreted less C-peptide(P<0.05) and maintained a higher level of blood glucose concentration (P<0.01) after the operation. There was no significant difference in insulin concentrations between the two groups. The level of blood glucose beyond the first 2 weeks after drug withdrawal in group FK506+MMF+Pred decreased obviously (P<0.05), and the secretion of insulin and C-peptide increased. These results were compared with those the first 2 weeks after transplantation and the first 2 weeks after drug withdrawal. CONCLUSIONS: Both regimens of FK506+MMF and FK506+MMF+Pred could provide effective immunosup-pression. Moreover the combined glucocorticoid-free immunosuppressive strategy of low-dose FK506 and MMF could protect islet grafts in islet transplantation without diabetogenic side-effects.

Islet transplantation in multicenter networks:the GRAGIL example

Purpose of review The enthusiasm generated by the results of the Edmonton protocol of islet transplantation is inciting a great number of institutions to start such programs.However,the procedure of islet isolation and purification is costly,complex and technically challenging.In order to share costs and to avoid facing the steep learning curve of the procedure,many centers interested in islet transplantation have looked into collaborating with experienced groups serving as core islet isolation facilities.Recent findings The proof of principle that remote islet processing and shipment could be successfully implemented with obtainng the Portland/Minneapolis,Huddinge/Giessen and Houston/Miami partnerships.Moreover,in order to increase both the donor pool and the number of patients gaining access to islet transplantation,multicenter networks,such as the Swiss-French GRAGIL consortium and the 4-country Nordic Network in Scandinavia have been built.The GRAGIL group has been fully operational since 1999,allowing the transplantation of 27 islet preparations processed in Geneva,Switzerland into 20 recipients in France over the course of 4.5 years.Organizational issues in the design of such networks are discussed based on the example of the GRAGIL experience.Summary The feasibility and the efficiency of islet transplantation in multicenter networks have been demonstrated.This strategy allows to increase the donor pool and the accessibility to islet transplantation in an extended population area.(J Intervent Radiol,2006,15: 626-631)

Development of microfluidic devices for islet transplantation and islet physiologys

本文讨论了芝加哥伊利诺伊大学研发的几种用于人胰岛生理和病理生理学研究的微流体装置及其在人胰岛移植中的应用。首先介绍了作为l型糖尿病临床治疗的胰岛移植领域关键问题。随后,回顾了可以解决这些关键问题的微流体装置、每一种微流体装置的独特之处及他们的应用。此外,本文也简单地讨论了芝加哥伊利诺伊大学这几种微流体装置的设计和制造原理。

全胰切除联合自体胰岛细胞移植的研究进展

自20世纪70年代以来,慢性胰腺炎患者已从全胰切除术合并自体胰岛细胞移植中获益。在过去的几十年里,随着外科技术和围手术期管理的不断改进,胰岛细胞功能、胰岛素不依赖率和患者存活率不断得到提高。本文归纳总结了自体胰岛细胞移植的术前适应证、手术操作的发展过程、术后管理监测的把控以及预后评估方面的研究进展。

黄芪多糖保护胰岛β细胞改善大鼠2型糖尿病

目的探讨黄芪多糖(APS)对2型糖尿病(T2DM)大鼠胰岛β细胞功能和数量的影响。方法 SD大鼠随机分为正常对照组、T2DM模型组和APS治疗组,每组8只。T2DM模型组大鼠采用高脂饮食联合链脲佐菌素构建T2DM模型,APS治疗组给予APS治疗(每天700mg/kg,APS含量70%)。药物干预8周后处死大鼠,取血测量大鼠的空腹血糖(FBG)、三酰甘油(TG)、总胆固醇(TC)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)以及空腹胰岛素(FINS)水平,并计算胰岛素分泌指数HOMA-β;取胰腺组织用苏木精-伊红染色观察组织病理学特征,并采用免疫组化法观察并计数胰岛β细胞。结果 (1)与正常对照组比较,T2DM模型组大鼠的FBG、TG和LDL-C升高,HDL-C、FINS和HOMA-β降低(P<0.05);与T2DM模型组比较,APS治疗组大鼠的FBG、TG和LDL-C降低(P<0.05),FINS和HOMA-β升高(P<0.05)。(2)与正常对照组比较,T2DM模型组大鼠的胰岛萎缩,伴有颗粒脱失及空泡变性现象,并且胰岛内β细胞的数量减少(P<0.05);与T2DM模型组比较,APS治疗组大鼠的胰岛体积增大,颗粒脱失和空泡变性现象有所改善,胰岛内β细胞的数量增加(P<0.05)。结论 APS能够改善T2DM大鼠的糖脂代谢,其机制可能是通过保护T2DM大鼠胰岛β细胞,进而促进胰岛素的分泌来实现的。

高浓度葡萄糖对胰岛细胞凋亡及凋亡相关基因的影响

目的:观察高浓度葡萄糖体外对胰岛细胞凋亡及凋亡相关基因表达的影响,探讨葡萄糖毒性及其分子机制。方法:应用TUNEL法检测高浓度葡萄糖培养后大鼠胰岛细胞和小鼠βTc3细胞凋亡百分率;应用定量RT-PCR(QRT-PCR)检测培养后大鼠胰岛细胞和小鼠βTc3细胞bcl-2和baxmRNA的表达;应用定量RT-PCR检测低剂量链脲佐菌素糖尿病大鼠胰岛bcl-2和baxmRNA的表达。结果:高浓度葡萄糖使原代培养的大鼠胰岛细胞和βTc-3的凋亡细胞比例明显增加;胰岛细胞凋亡过程中,诱导凋亡基因bax的mRNA表达水平明显升高,抵抗凋亡基因bcl-2mRNA表达水平明显下降,致使bcl-2/bax比率明显降低;对低剂量链脲佐菌素糖尿病大鼠胰岛凋亡相关基因QRT-PCR检测也显示同样的结果。结论:高浓度葡萄糖可能通过诱导胰岛细胞凋亡增加而加重糖尿病,其中bcl-2/baxmRNA表达比率变化可能起重要作用。

盐酸小檗碱对链脲霉素致大鼠离体胰岛B细胞损伤的影响

目的 :探讨盐酸小檗碱 (Ber-HCl)对链脲霉素 (STZ)致大鼠离体胰岛B细胞损伤的影响。方法 :用STZ(3mmol/L)造成大鼠胰岛B细胞损伤模型 ,采用新生大鼠胰岛离体培养方法 ,通过放免检测培养液中基础胰岛素分泌 (BIS)及 1h高浓度葡萄糖刺激胰岛素释放 (HGSIR)水平 ,判断Ber -HCl对STZ致离体胰岛B细胞损伤的影响。结果 :各浓度 (10、2 0、4 0、80 μmol/L)Ber -HCl组的BIS与单纯STZ组相比无明显差异 (P >0 .0 5 ) ,且均低于正常对照组 (P <0 .0 1) ;4 0、80 μmol/LBer-HCl组的HGSIR高于单纯STZ组 (P <0 .0 5 ) ,各浓度Ber-HCl组和单纯STZ组的HGSIR又均明显低于正常对照组 (P <0 .0 5 ,P <0 .0 1)。结论 :Ber

高浓度游离脂肪酸对胰岛细胞凋亡及相关基因表达的影响

目的 :观察高浓度游离脂肪酸体外对胰岛细胞凋亡和凋亡相关基因表达的影响。方法 :应用TUNEL法检测高浓度游离脂肪酸培养后大鼠胰岛细胞和小鼠 βTc3细胞凋亡百分率 ,应用定量RT -PCR(QRT -PCR)检测培养后bcl-2、bax、c-myc、p53和fasmRNA的表达。结果 :高浓度游离脂肪酸使原代培养的大鼠胰岛细胞和βTc3的凋亡细胞比例明显增加。胰岛细胞凋亡过程中 ,诱导凋亡基因bax、c -myc、fas的mRNA表达水平明显升高 ,抵抗凋亡基因bcl-2mRNA表达水平明显下降 ,p53mRNA表达水平无明显改变。结论 :游离脂肪酸可能通过诱导胰岛细胞凋亡导致或加重糖尿病 。

体外诱导大鼠骨髓间质干细胞分化为胰岛素分泌细胞(英文)

目的探索大鼠骨髓间质干细胞体外诱导分化为胰岛素分泌细胞的方法。为解决胰岛移植物来源匮乏这一问题提供新的思路。方法 采用横向分化技术,将成年大鼠骨髓间质干细胞诱导成为胰岛素分泌细胞。间接免疫荧光法鉴定诱导前后细胞nestin、胰岛素、胰高血糖素、生长抑素及Pdx-1的表达,RT-PCR法检测诱导前后细胞nestin,胰岛素-1,葡萄糖转运子-2,葡萄糖激酶及其转录因子Isl-1,Pdx-1,Pax-4和Pax-6 mRNA的表达;测定24 h胰岛素分泌量和胰岛素刺激实验评价诱导前后细胞的功能。结果 诱导5 h,nestin阳性细胞为(44.6±7.3)％;诱导24 h,nestin阳性细胞增至(61.8±8.4)％;此后,nestin阳性细胞数目开始下降,诱导第14天后,nestin表达基本消失。诱导后的胰岛素分泌细胞可以表达胰岛素、胰高血糖素、生长抑素和Pdx-1等蛋白;表达胰岛素-1、葡萄糖转运子-2、葡萄糖激酶及其多种转录因子mRNA;胰岛素分泌量增加;胰岛素刺激实验反应敏感等。而诱导前MSCc不县备上述特点。结论 大鼠骨髓间质干细胞体外可以诱导成为胰岛素分泌细胞,为胰岛移植开辟新的研究思路。