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-immunological challenges and current perspectives

Pancreatic islet transplantation is a minimally invasive procedure aiming to reverse the effects of insulin deficiency in patients with type 1 diabetes(T1D)by transplanting pancreatic beta cells.Overall,pancreatic islet transplantation has improved to a great extent,and cellular replacement will likely become the mainstay treatment.We review pancreatic islet transplantation as a treatment for T1D and the immunological challenges faced.Published data demonstrated that the time for islet cell transfusion varied between 2 and 10 h.Approximately 54%of the patients gained insulin independence at the end of the first year,while only 20%remained insulin-free at the end of the second year.Eventually,most transplanted patients return to using some form of exogenous insulin within a few years after the transplantation,which imposed the need to improve immunological factors before transplantation.We also discuss the immunosuppressive regimens,apoptotic donor lymphocytes,anti-TIM-1 antibodies,mixed chimerism-based tolerance induction,induction of antigen-specific tolerance utilizing ethylene carbodiimide-fixed splenocytes,pretransplant infusions of donor apoptotic cells,B cell depletion,preconditioning of isolated islets,inducing local immunotolerance,cell encapsulation and immunoisolation,using of biomaterials,immunomodulatory cells,etc.

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。

WJSC 6^(th) Anniversary Special Issues(2):Mesenchymal stem cells Mesenchymal stem cells help pancreatic islet transplantation to control type 1 diabetes

Islet cell transplantation has therapeutic potential to treat type 1 diabetes,which is characterized by autoimmune destruction of insulin-producing pancreatic isletβcells.It represents a minimal invasive approach forβcell replacement,but long-term blood control is still largely unachievable.This phenomenon can be attributed to the lack of islet vasculature and hypoxic environment in the immediate post-transplantation period that contributes to the acute loss of islets by ischemia.Moreover,graft failures continue to occur because of immunological rejection,despite the use of potent immunosuppressive agents.Mesenchymal stem cells(MSCs)have the potential to enhance islet transplantation by suppressing inflammatory damage and immune mediated rejection.In this review we discuss the impact of MSCs on islet transplantation and focus on the potential role of MSCs in protecting islet grafts from early graft failure and from autoimmune attack.

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.

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.

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.

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.

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)

间充质干细胞在1型糖尿病胰岛移植中的应用进展

胰岛移植被认为是治疗1型糖尿病的有效方法之一,但其疗效受到多种因素限制。胰岛在分离、培养和移植过程中的缺氧、应激及排斥反应,都会影响胰岛移植的结局。间充质干细胞(MSC)因其抗炎、促进血管生成和调节免疫代谢等生物特性,一直备受研究者关注。此外,MSC的衍生物如外泌体在调节缺氧诱导的氧化应激、促进机体血管形成和调节免疫方面也具有重要作用。基于MSC的胰岛移植可能是1型糖尿病的有效治疗方法。因此,本文就MSC在胰岛移植前后发挥的潜在作用进行综述,并探讨其临床应用及局限性,以期为今后胰岛移植治疗1型糖尿病的相关研究提供新的思路和见解。

The effect of FasL gene transfer to islet cells on pancreatic islet allografts

OBJECTIVE: To investigate the effect of FasL gene transfer to islet cells on pancreatic islet allografts. METHODS: A recombinant and replication-deficient type-5 adenovirus encoding murine FasL (AdV- FasL) was constructed by the method of calcium phosphate precipitation. Pancreatic islets were infected with the recombinant adenovirus AdV-FasL, and transplanted into diabetic recipients. FasL expression was detected by RT-PCR and immunohistochemistry. The survival of allografts and the apoptosis of gene transferred islet allografts were analyzed. RESULTS: All animals receiving islet allograft alone returned to a diabetic state by several days (mean survival time 6.3±0.6 days). Compared with the control group, no delayed rejection and prolonged survival of allografts were observed in the group of FasL gene transfer. The rejection was accelerated and the allograft survival was shortened to 3.4±0.2 days (P<0.05). Pancreatic islets infected with AdV- FasL demonstrated positive staining of FasL at 24 h, with an increased intensity at 48 h, but not in AdV-5 infected or uninfected islets. TUNEL labeling of pancreatic islet allografts at 24, 48 h revealed apoptosis that was not in AdV-5 infected allografts. CONCLUSIONS: Though co-transplantation of FasL-expressing testicular cells can induce privilege of islet allografts and prolong allograft survival, direct expression of FasL on islet allografts infected with AdV-FasL may accelerate islets rejection by islet apoptosis and granulocyte infiltration.

减低剂量预处理单倍体外周血造血干细胞移植术后早期免疫重建的临床研究

目的分析新疆医科大学第一附属医院血液病中心制定的减低剂量预处理高剂量非体外去T细胞单倍体外周血造血干细胞移植术(RIC-haplo-HDPSCT)后早期细胞免疫及体液免疫的动态变化特点。方法以行RIC-haplo-HDPSCT移植术的54例患者和20例健康者(对照组)为研究对象,通过流式细胞法检测患者移植术前、移植术后30 d、60 d、90 d及对照组外周血中CD3、CD4、CD8、CD19、CD56细胞的百分比,ELISA法检测移植后30 d、60 d、90 d患者免疫细胞(IgG、IgA、IgM)的百分比,观察RIC-haplo-HDPSCT术后上述免疫细胞的动态变化规律,并比较发生急性移植物抗宿主病(aGVHD)与未发生aGVHD患者各免疫细胞百分比的变化情况。结果(1)在此移植模式下,CD3^(+)T细胞在术后先下降后升高,至移植术后60 d可达对照组水平,60 d后缓慢降低,与对照组相比无差异;CD4^(+)T细胞在移植术后30 d显著下降,随后逐渐恢复,但在术后90 d内均低于对照组;CD8^(+)T细胞在移植术后先降低后显著上升,在移植术后60 d上升至高于对照组水平,之后缓慢下降,与对照组相比无差异;CD19^(+)B细胞在移植术后缓慢上升,但移植术后早期仍明显低于对照组。CD56^(+)NK细胞移植术后30 d时显著高于对照组,30 d后呈下降趋势,与对照组相比无差异。IgG在移植术后30 d升高,然后逐步下降,与对照组相比,差异无统计学意义。IgA及IgM移植术后早期低于对照组,但差异无统计学意义。(2)发生aGVHD患者的CD8^(+)T细胞数量在移植术后30 d时明显高于未发生aGVHD患者。CD56^(+)NK细胞数量在移植术后30 d明显低于未发生aGVHD患者。结论本中心制定的RIC-haplo-HDPSCT移植模式术后NK细胞恢复最快,其次为CD8^(+)T细胞及CD3^(+)T细胞;各体液免疫细胞在移植术后呈下降趋势,但降低并不明显,CD8^(+)T细胞数量增多及CD56^(+)NK细胞的减少可能与aGVHD的发生有关。

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。

间充质干细胞联合骨髓细胞输注诱导胰岛移植免疫耐受的研究

目的观察间充质干细胞(MSC)联合骨髓细胞(BMC)输注对同种异体小鼠胰岛移植嵌合状态及胰岛移植物存活时间的影响。方法C57BL/6小鼠和BALB/C小鼠分别作为供、受体。应用链脲佐菌素制备BALB/C小鼠糖尿病模型,将分离纯化的C57BL/6小鼠胰岛移植到上述糖尿病模型小鼠的肾包囊下,用抗CD154单抗进行受体预处理。将接受胰岛移植的25只BALB/C受体鼠随机分为A组(单纯胰岛移植);B组(供体MSC悬液0.5ml输注);C组(供体BMC悬液0.5ml输注);D组(供体BMC和MSC各0.5ml输注);E组(供体BMC和第三品系的KM小鼠MSC各0.5ml输注),每组5只,所有细胞在胰岛移植后经尾静脉输注。比较以上各组供体细胞嵌合率(DC)和胰岛移植物存活时间的差异。结果在胰岛移植后第30天,MSC联合BMC输注的D、E两组与单纯BMC输注的C组比较,其DC显著升高(P<0.01);胰岛存活时间亦显著延长[(77.0±7.7d)和(61.0±2.2d)vs(53.0±16.4d)(P<0.01)]。胰岛移植后第60天,D组与E组比较,DC维持在更高水平,且胰岛移植物存活时间更长(P<0.05)。结论MSC联合BMC输注比单纯BMC输注能够维持更长时间的混合嵌合状态,并延长胰岛移植物存活时间;供体来源的MSC输注比非供体来源的MSC输注更有效。

胰岛移植用免疫隔离微胶囊的牢固度、生物相容性和通透性研究

目的 :为了探讨用于胰岛移植的海藻酸钠 聚赖氨酸 海藻酸钠 (APA)生物膜的免疫隔离效果。方法 :采用高压静电成囊装置 ,制备APA微胶囊和微囊化胰岛 ,微囊直径为 0 2 5～ 0 5 5mm ;取空微囊 ,利用恒温振荡仪振荡后测定其破损率 ;将空微囊移植至小鼠腹腔 ,分别于不同时间由腹腔中灌洗出微胶囊 ,记数并观察其形态 ;取一定量空微囊分别与IgG、BSA和胰蛋白酶孵育 ,测量其浓度变化确定APA微胶囊的通透性。结果 :采用高压静电成囊技术制成的APA微胶囊呈球形 ,大小均匀、表面光滑 ,具有较好的生物相容性 ;粒径为 0 2 5～ 0 35mm的微胶囊其牢固度大于粒径为 0 45～ 0 5 5mm的微胶囊 ;葡萄糖、胰岛素等小分子物质能够自由通过微囊膜 ,胰蛋白酶也可通过 ,但速度较慢 ;大分子物质牛血清白蛋白和免疫球蛋白则不能透入APA微囊。结论 :采用高压静电成囊技术可制备高质量粒径为 0 2 5～ 0 35mm的微胶囊 ;这是具有良好免疫隔离性能的APA微胶囊。

免疫缺陷树突状细胞诱导异种胰岛细胞移植耐受

目的 研究受体来源免疫缺陷树突状细胞(dendriticcell, DC)诱导异种胰岛细胞移植的免疫耐受作用及其机制。方法 从BALB/C小鼠骨髓干细胞诱导分化免疫缺陷DC,负载Wistar大鼠MHC抗原。将上述DC通过尾静脉回输糖尿病小鼠体内(预处理组), 7d后分别将Wistar或SD大鼠胰岛细胞移植于受体鼠肾包膜下。观察移植物存活时间,检测T细胞增殖及TH1 /TH2细胞因子的表达。结果 与对照组相比,预处理组胰岛细胞存活时间明显延长(P < 0. 0 5 ),而以SD大鼠胰岛细胞作为供体的移植物存活时间无明显改变。免疫缺陷DC预处理受体鼠T细胞增殖反应微弱,且TH1 /TH2细胞因子表达明显下降。结论 负载异种MHC抗原的免疫缺陷型DC预处理受体可诱导抗原特异性T细胞无能,以及TH1 /TH2细胞因子的低表达,从而有效地延长异种胰岛细胞存活时间。

我国首例成人胰岛细胞肝内移植术的观察与护理

目的总结成人胰岛细胞肝内移植治疗1型糖尿病的护理经验。方法2003年间对一例1型糖尿病患者进行了3次成人胰岛细胞肝内移植术,术后3年来连续观察移植前后的饮食、血糖、C肽、胰岛素用量和免疫抑制剂的应用等指标,应用各种护理方法,密切观察病情变化和各种并发症。结果移植后患者饮食不受限制,生长发育良好,胰岛素用量减少到移植前的25%,切口愈合良好,心理健康,掌握了术后自我管理的方法,总结出了个体化的护理经验。结论建立起了成人胰岛细胞肝内移植的一整套护理方法,取得了治疗1型糖尿病的临床疗效。

免疫抑制剂对人胰岛细胞的毒性作用

目的探讨临床常用免疫抑制剂对人胰岛细胞的直接毒性作用。方法应用体外分离纯化后培养的人胰岛细胞,以刺激系数和丫啶橙/碘丙啶染色为效应指标,检验低、中、高不同浓度免疫抑制剂对人胰岛细胞的直接毒性作用。结果体外培养3天后,低浓度免疫抑制剂未表现出胰岛毒性作用;中浓度免疫抑制剂对胰岛的毒性不同,以他克莫司为重;高浓度免疫抑制剂有较强的胰岛毒性。结论免疫抑制剂对人胰岛细胞直接毒性作用与浓度相关,选择低胰岛毒性免疫抑制方案有助于提高胰岛移植成功率。

不同体重供体及理化因素对小鼠胰岛分离的影响

目的:探讨提高小鼠胰岛数量和质量的可靠方法。方法:采用胆管内胶原酶灌注和Histopaque1077密度离心法分离纯化胰岛。分别观察供体的体重、胶原酶的浓度、消化的时间及温度对胰岛分离起的作用。在体外,ELISA检测葡萄糖刺激引起的小鼠胰岛素分泌。在体内,胰岛移植评价胰岛对糖尿病小鼠血糖的调节功能。结果:高体重供体组胰岛的收获量显著高于低体重供体组(P<0.01)。胶原酶浓度、消化时间对胰岛收获量起重要作用。胰岛活率≥95%。体外胰岛素释放试验表明胰岛功能良好。同种异体胰岛移植的糖尿病小鼠高血糖状态得到逆转。结论:小鼠的体重、胶原酶的浓度、消化时间和温度是影响胰岛数量和功能的重要因素。

M2型巨噬细胞在糖尿病小鼠同种异体胰岛移植中的抗排斥作用

目的:探讨腹膜腔来源的M2型巨噬细胞对糖尿病小鼠同种异体胰岛移植的抗排斥作用。方法:体外分离诱导C57BL/6小鼠腹膜腔M2型和M0型细胞,流式细胞术鉴定巨噬细胞表型。链脲佐菌素(streptozotocin,STZ)诱导C57BL/6雄性小鼠的糖尿病模型作为受体,分离BALB/c小鼠胰岛细胞移植于C57BL/6糖尿病小鼠左肾包膜下。将移植后糖尿病小鼠随机分为3组,每组8只。于胰岛移植术后1,3,7 d分别经尾静脉输注PBS(islet+PBS组)、2.5×106个M0型巨噬细胞(islet+M0组),2.5×106个M2型巨噬细胞(islet+M2组)。移植术后取尾静脉血监测受体血糖水平的变化,记录胰岛存活时间,并于移植后10 d每组随机抽取2只小鼠处死取左肾做病理学检查,观察移植物形态结构和胰岛素表达水平。结果:Islet+PBS组、islet+M0组和islet+M2组移植物的中位存活时间分别为6.5(4~10),7.5(4~10)和24(﹥15)d;与islet+PBS组和islet+M0组比较,islet+M2组移植物调节血糖正常水平时间和中位存活时间明显延长(P<0.01)。病理学检查结果显示islet+M2组胰岛移植10 d后移植物结构完整,胰岛素染色阳性;而islet+PBS组和islet+M0组胰岛移植物均被排斥,胰岛素染色阴性,局部见大量淋巴细胞浸润。结论:腹膜腔来源的M2型巨噬细胞能减轻受体对胰岛移植物的免疫排斥反应,延长胰岛移植物的存活时间,从而提高受体对血糖的耐受能力。