胰岛移植术后立即经血液介导的炎症反应机制

目的初步探讨胰岛移植术后立即经血液介导的炎症反应(instant blood-mediated in-flammatory reaction,IBMIR)的发生机制。方法Wistar大鼠,雌雄不限,体重为250～300g,分离、纯化胰岛,建立体外循环模型,于37℃下循环反应60min后加入胰岛800当量模拟IBMIR,分别于循环前、加入胰岛前、加入胰岛后5min、15min、30min、60min后留取血液行血常规检测,用酶标法检测血浆凝血酶-抗凝血酶复合物(thrombin-antithrombin complex,TAT),并用酶联免疫吸附法检查血浆补体3a(C3a)含量。循环60min后取过滤残余血栓和组织行形态学检查。结果加入胰岛体外循环60min后血液中的血小板几乎全部耗竭,白细胞及单核细胞减少亦较为明显,但淋巴细胞比例变化不明显。血浆中的TAT、C3a含量随循环时间延长而增加。循环60min后的过滤残余的血栓块和组织中胰岛数量较少,结构破坏严重,被膜不完整,胰岛周围被微血栓包绕,大量中性粒细胞浸润。结论凝血反应、补体激活和白细胞激活可能是导致IBMIR的重要机制。     

胰岛细胞移植后发生立即经血液介导的炎症反应的机制

在胰岛细胞移植的过程中,当其一接触受体血液即会引发立即经血液介导的炎症反应(IBMIR),使胰岛细胞迅速破坏失去分泌胰岛素的功能,笔者就IBMIR发生机制所涉及的问题,包括凝血系统的激活、补体系统的激活和抗体的作用、炎症介质和炎症细胞的浸润以及葡萄糖毒性的作用等作一综述。     

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。     

阿加曲班联合尿激酶治疗下肢深静脉血栓形成的临床研究

目的 探讨阿加曲班合尿激酶治疗下肢深静脉血栓（DVT)的临床应用价值。方法 选择我科2006年1月～2010年12月收治的160例下肢DVT患者,随机分为阿加曲班组和对照组,各组80例。阿加曲班组患者给予阿加曲班注射液和尿激酶治疗,对照组患者予低分子肝素钙合尿激酶治疗,共治疗2周。比较两组患者临床疗效的差异。结果 阿加曲班组临床疗效优于对照组（P＜0.05）,两组患者凝血指标(PT、TT和APTT) 及PLT变化均处于正常范围。结论 阿加曲班合尿激酶治疗下肢DVT疗效肯定,副作用小,安全性大,值得临床推广。     

阿加曲班与肝素在血液透析抗凝治疗中作用的比较分析

目的：探讨阿加曲班（argatroban）在血液透析（hemodialysis,HD）抗凝治疗中作用的疗效及安全性.方法：将90例次HD患者随机分成实验组和对照组,实验组45例次,使用阿加曲班抗凝,对照组45例次,使用肝素抗凝,监测HD患者治疗前血路管动脉端、治疗中2 h、治疗结束前血路管动、静脉端及治疗后1 h活化部分凝血活酶时间（APTT）,治疗过程中管路和透析器凝血情况,治疗后穿刺点压迫止血平均时间及组织器官24 h内出血情况（包括牙龈出血、鼻衄、结膜出血、皮下出血点、血尿、黑便及便血等）.结果：实验组HD治疗中APTT明显延长,达到HD抗凝治疗要求,与HD治疗前比较差异有统计学意义（P＜0.01）;实验组与对照组治疗中APTT比较差异有统计学意义（P＜0.01）;实验组治疗后1 h APTT恢复基本正常,而对照组仍然很高,两组比较差异有统计学意义（P＜0.01）;实验组与对照组治疗中管路和透析器凝血情况比较差别不大,两组比较差异无统计学意义（P＞0.05）;实验组与对照组治疗后穿刺点压迫止血平均时间比较差异有统计学意义（P＜0.05）;实验组治疗后组织器官出血较对照组明显减少,两组比较差异有统计学意义（P ＜0.01）.结论：阿加曲班在HD抗凝治疗中与普通肝素效果相当,但出血风险少,安全性高,适用于有出血倾向的HD患者.     

输注胰岛抗原特异性Treg细胞延长同系NOD小鼠移植胰岛的存活时间

目的 探讨输注胰岛抗原特异性调节性T淋巴细胞(Treg细胞)对非肥胖糖尿病(NOD)小鼠同系胰岛移植物存活时间的影响.方法·以未成熟树突状细胞(imDC)联合谷氨酸脱羧酶-65在体外诱导童贞T淋巴细胞分化成胰岛抗原特异性Treg细胞.以已发生糖尿病的NOD小鼠为受者,将分离得到的尚未进展为糖尿病的NOD小鼠的胰岛(500胰岛当量)移植至受者的肾包膜下,对照组不行移植,只观察血糖变化;单纯胰岛移植组只进行胰岛移植,不输注胰岛抗原特异性Treg细胞;实验组于术前1d静脉输注1×106个胰岛抗原特异性Treg细胞,然后进行胰岛移植.术后检测受者的血糖,以判断移植胰岛的存活时间,观察胰岛移植物的病理学变化.结果 对照组血糖持续高于11.1 mmol/L;单纯胰岛移植组小鼠的血糖于术后1～2 d降至正常,到7～17d时开始陆续升高,并维持在术前水平,移植物存活时间为(12.2±2.6)d;实验组小鼠的血糖于术后1～2 d降至正常,至第27天开始有小鼠血糖升高超过11.1 mmol/L,第43天时,所有小鼠的血糖均超过11.1mmol/L,移植物的存活时间为(35.2±4.3)d,明显长于单纯胰岛移植组(P＜0.01).单纯胰岛移植组的移植胰岛有明显的淋巴细胞浸润,并伴有胰岛细胞严重破坏,胰岛素染色未见完整的胰岛存在,仅有极少量残存的分泌胰岛素的胰岛细胞;实验组第15天时移植胰岛形态完整,仅有少量淋巴细胞浸润,分泌胰岛素的胰岛大量存在.结论 体外诱导产生的胰岛抗原特异性Treg细胞可以延缓自身免疫系统对移植胰岛的破坏,明显延长NOD小鼠移植胰岛的存活时间.     

RNA干扰技术抑制诱导型一氧化氮合酶基因表达对大鼠胰岛细胞凋亡和功能的影响

目的 探讨诱导型一氧化氮合酶(iNOS)基因在大鼠胰岛细胞培养过程中对胰岛细胞凋亡和胰岛素分泌的影响及其机制.方法 从30只Wistar大鼠获得胰岛,随机分为5组.大鼠胰岛体外培养中,使用针对iNOS的siRNA转染胰岛.根据RNA干扰(RNAi)条件以及培养液中是否加入细胞因子TNF-α和IL-1β将胰岛分为5组:空白对照组、细胞因子组、阴性对照组、RNAi组以及RNAi+细胞因子组.通过RT-PCR和Western blot检测RNAi效果,RT-PCR和TUNNEL法检测胰岛凋亡情况,胰岛素释放实验检测胰岛功能.结果 每只大鼠可获得500～600 IEQ胰岛.RNAi可以沉默大鼠胰岛组织iNOS基因,抑制iNOS生成.胰岛经细胞因子IL-1β和TNF-α处理后,促凋亡基因Bax和Fas表达明显升高,胰岛素释放减少.经RNAi抑制iNOS基因后,胰岛与细胞因子IL-1β和TNF-α共同培养时,促凋亡基因表达明显下降,凋亡细胞减少,胰岛素释放增加.结论 RNAi技术抑制胰岛iNOS基因表达后,可减少胰岛细胞凋亡,改善胰岛的存活和功能.     

提高胚胎胰岛冷冻保存质量的实验研究

目的：研究三维组织细胞旋转培养系统（RCCS）对胚胎胰岛冷冻保存复苏后质量的影响。方法：将胚胎胰岛平均分为实验组1、2和对照组,实验组1、2冷冻保存胰岛,冷冻保存前后分别用RCCS培养和普通培养,对照组经RCCS培养新鲜胰岛。切取胚胎胰腺,经胶原酶V消化、纯化,培养3d,然后进行标准冷冻保存,复苏后继续培养,并检测各组胰岛数量及活性。结果：纯化后每个胚胎收获胰岛最多5115.43IEQ,最少2331.98IEQ,平均（3551.27&#177;253.76）IEQ。实验组1胰岛细胞存活率、胰岛素释放量、胰岛素刺激指数均高于实验组2。结论：RCCS培养有利于胰岛细胞的生长繁殖,使其具有更好的分泌胰岛素能力,该方法同胰岛冷冻保存相结合,可以进一步提高胰岛的冷冻保存效果,为胰岛库的成功建立探索出一条新的途径。     

阿加曲班治疗下肢深静脉血栓的疗效评价

目的比较常规使用肝素和阿加曲班治疗下肢深静脉血栓（DVT）患者的临床疗效。方法将188例下肢DVT患者按照随机数字表分成阿加曲班组（n=94）和对照组（低分子肝素钙＋尿激酶,n=94）,比较2组患者治疗前、后双侧肢体周径差和疗效的差异,并在治疗过程中监测凝血指标（PT、APTT及PLT）变化。结果阿加曲班组治疗10d后,双侧肢体周径差较治疗前明显减小（P〈0.05）,总有效率（97.87%）优于对照组（89.37%）,P〈0.05。阿加曲班组无血小板减少症（HIT）发生,对照组发生2例HIT;阿加曲班组PT、APTT和PLT变化均处于正常范围,与对照组比较差异无统计学意义（P〉0.05）。结论阿加曲班治疗下肢DVT安全、有效。     

转化生长因子-β1对大鼠同种胰岛移植物的影响

目的　研究转化生长因子 β1 (TGF β1 )对大鼠同种胰岛移植物体内外的内分泌功能及存活的影响。方法　构建TGF β1的真核表达载体 pcDNA3 TGF ,并将其转染纯化后的Wistar大鼠胰岛 ,逆转录 聚合酶链式反应 (RT PCR)检测TGF β1和大鼠胰岛素 (Ins1 )在实验组和对照组胰岛中的表达 ,放射免疫法 (RIA)检测体外培养液中的胰岛素含量 ;链脲霉素 (STZ) 60mg/kg体重腹腔内注射制作SD大鼠模型 ,分别将实验组和空白对照组Wistar胰岛移植到受体SD大鼠的肾包囊下 ,观察其在体内逆转糖尿病的作用以及作用时间的长短 ,并检测受体大鼠的糖耐量。结果　实验组胰岛有TGF β1和Ins1的表达 ,而对照组仅有Ins1的表达 ;实验组体外培养液中的胰岛素含量在基础相和刺激相分别为 (3 .86± 1 .2 4 ) μg/L和 (8.43± 1 .59) μg/L ,对照组体外培养液中的胰岛素含量在基础相和刺激相分别为 (4.1 3± 1 .45) μg/L和 (8.95± 1 .74) μg/L ,组间差异无显著性 (P>0 .0 5) ,而组内差异有显著性 (P <0 .0 5) ;实验组平均正常血糖维持时间 (2 6 .8± 2 .9)d比对照组的 (1 3 .2± 4 .5)d显著延长。对照组糖耐量曲线均明显高于实验组。结论　TGF β1对胰岛移植物的内分泌功能无损害作用 ,但是可以延长胰岛移植物体内存活时间     

Tissue factor knockdown in porcine islets: an effective approach to suppressing the instant blood-mediated inflammatory reaction

Tissue factor (TF) expression on islets has been shown to trigger instant blood-mediated inflammatory reaction (IBMIR), leading to rapid islet loss in portal vein islet transplantation. This study investigated whether antisense RNA-mediated TF gene knockdown in islets could suppress IBMIR as a strategy to overcome IBMIR. Neonatal porcine islet cell clusters (NICCs) were transfected with or without TF-specific antisense RNA or a nonspecific RNA by a lipid-based method. Expression of both TF gene and protein in NICCs was analyzed after transfection by real-time PCR, Western blot, and FACS, respectively. The impact of antisense RNA transfection on NICC viability and in vitro function was examined by FACS and insulin release test, respectively. The effect of TF knockdown in NICCs on IBMIR was assessed with an in vitro tubing loop assay using human blood. A significant reduction in TF gene and protein expression was achieved in TF antisense RNA but not control RNA transfected NICCs, which did not affect NICCs' viability or their insulin secreting capacity. Incubation of TF antisense RNA transfected with human blood resulted in a considerable reduction in blood clot formation, platelet consumption, and complement and coagulation activation compared to that observed in the loops containing human blood and untreated or control RNA transfected NICCs. Consistent with these findings, infiltrating neutrophils in the blood clots with entrapped TF antisense RNA transfected NICCs was also reduced substantially compared to that seen in the clots containing untreated or control RNA transfected NICCs. This study presents a nontoxic TF antisense RNA-mediated TF knockdown in porcine islets that leads to an effective suppression of IBMIR, suggesting a potentially new strategy to improve islet transplantation outcomes.     

Composite Islet-Endothelial Cell Grafts: A Novel Approach to Counteract Innate Immunity in Islet Transplantation

An instant blood-mediated inflammatory reaction (IBMIR) is elicited when islets come in contact with blood after intraportal transplantation. In contrast, endothelial cells (EC) readily tolerate contact with blood. A conceivable strategy to overcome IBMIR would be to create composite islet-EC grafts. Human islets were co-cultured with primary human aortic endothelial cells (HAEC) for 2-7 days to obtain 50-90% coverage. HAEC-coated islets were exposed to ABO-identical blood and analyzed with regard to clotting time, signs of inflammation and cell infiltration. Composite islet-HAEC graft survival was assessed after transplantation to athymic (nu/nu) nude mice. Exposed to blood, HAEC-coated islets induced less activation of coagulation and complement compared to control islets. Also, platelet and leukocyte consumption in blood was decreased. Clots with entrapped HAEC-coated islets showed less infiltration of CD11b+ cells. The extent of protection correlated to the level of HAEC coverage. Transplanted composite grafts stained positive for insulin and PECAM-1 demonstrating presence of both islets and HAEC within the islet graft 7 weeks after transplantation. Composite islet-HAEC grafts reduce all components of IBMIR. Refinement of the technique will allow introduction of composite islet-EC grafts in clinical islet transplantation, using autologous EC expanded in vitro and kept frozen until allogeneic islets become available for that specific recipient.     

Immune checkpoint CD47 molecule engineered islets mitigate instant blood‐mediated inflammatory reaction and show improved engraftment following intraportal transplantation

Instant blood‐mediated inflammatory reaction (IBMIR) causes significant destruction of islets transplanted intraportally. Myeloid cells are a major culprit of IBMIR. Given the critical role of CD47 as a negative checkpoint for myeloid cells, we hypothesized that the presence of CD47 on islets will minimize graft loss by mitigating IBMIR. We herein report the generation of a chimeric construct, SA‐CD47, encompassing the extracellular domain of CD47 modified to include core streptavidin (SA). SA‐CD47 protein was expressed in insect cells and efficiently displayed on biotin‐modified mouse islet surface without a negative impact on their viability and function. Rat cells engineered with SA‐CD47 were refractory to phagocytosis by mouse macrophages. SA‐CD47‐engineered islets showed intact structure and minimal infiltration by CD11b⁺ granulocytes/macrophages as compared with SA‐engineered controls in an in vitro loop assay mitigating IBMIR. In a syngeneic marginal mass model of intraportal transplantation, SA‐CD47‐engineered islets showed better engraftment and function as compared with the SA‐control group (87.5% vs 14.3%). Engraftment was associated with low levels of intrahepatic inflammatory cells and mediators of islet destruction, including high‐mobility group box‐1, tissue factor, and IL‐1β. These findings support the use of CD47 as an innate immune checkpoint to mitigate IBMIR for enhanced islet engraftment with translational potential.     

Reduction of early graft loss after intraportal porcine islet transplantation in monkeys

BACKGROUND: Pig islets constitute a possible resolution to the shortage of human islets for transplantation. After intraportal infusion of porcine islets in primates, many islets are lost through what has been termed the instant blood-mediated inflammatory reaction (IBMIR). We report on our experience with IBMIR. METHODS: Ten monkeys underwent intraportal porcine islet transplantation. Immunosuppressive therapy was with conventional agents (n=3) or based on costimulation blockade (n=7). Treatment specific for IBMIR was administered in eight monkeys; two additional monkeys received no such therapy (group 1). Cobra venom factor completely inhibited complement activity in four (group 2) and dextran sulfate provided anticoagulation in four (group 3). Islet graft function was monitored by following blood glucose, insulin requirement, and porcine C-peptide values. RESULTS: In monkeys that received neither cobra venom factor nor dextran sulfate (group 1), there was rapid destruction of islets indicated by severe hypoglycemia and the need for dextrose infusion; C-peptide levels were initially low and further reduction occurred within the first five days. In both groups 2 and 3, there was significantly less destruction of islets and some reversal of diabetes. However, when 40,000 IEQ/kg were infused, normoglycemia was lost within five days, but when 80,000 IEQ/kg were infused in one case, normoglycemia was more persistent. We observed that even when C-peptide levels were in the normal range for healthy nondiabetic pigs, these were not sufficient to maintain normoglycemia in the monkeys. CONCLUSIONS: Although pretransplantation complement depletion or anticoagulation reduces porcine islet xenograft loss significantly, neither alone is sufficient to prevent IBMIR.     

Tissue factor produced by the endocrine cells of the islets of Langerhans is associated with a negative outcome of clinical islet transplantation

There are strong indications that only a small fraction of grafts successfully engraft in clinical islet transplantation. One explanation may be the instant blood-mediated inflammatory reaction (IBMIR) elicited by tissue factor, which is produced by the endocrine cells. In the present study, we show that islets intended for islet transplantation produce tissue factor in both the transmembrane and the alternatively spliced form and that the membrane-bound form is released as microparticles often associated with both insulin and glucagon granules. A low-molecular mass factor VIIa (FVIIa) inhibitor that indirectly blocks both forms of tissue factor was shown in vitro to be a promising drug to eliminate the IBMIR. Thrombin-antithrombin complex (TAT) and FVIIa-antithrombin complex (FVIIa-AT) were measured in nine patients who together received 20 infusions of isolated human islets. Both the TAT and FVIIa-AT complexes increased rapidly within 15-60 min after infusion. When the initial TAT and FVIIa-AT levels were plotted against the increase in C-peptide concentration after 7 days, patients with an initially strong IBMIR showed no significant increase in insulin synthesis after 7 days. In conclusion, tissue factor present in both the islets and the culture medium and elicits IBMIR, which affects the function of the transplanted islets.     

Dual Islet Transplantation Modeling of the Instant Blood‐Mediated Inflammatory Reaction

Islet xenotransplantation is a potential treatment for diabetes without the limitations of tissue availability. Although successful experimentally, early islet loss remains substantial and attributed to an instant blood‐mediated inflammatory reaction (IBMIR). This syndrome of islet destruction has been incompletely defined and characterization in pig‐to‐primate models has been hampered by logistical and statistical limitations of large animal studies. To further investigate IBMIR, we developed a novel in vivo dual islet transplant model to precisely characterize IBMIR as proof‐of‐concept that this model can serve to properly control experiments comparing modified xenoislet preparations. WT and α1,3‐galactosyltransferase knockout (GTKO) neonatal porcine islets were studied in nonimmunosuppressed rhesus macaques. Inert polyethylene microspheres served as a control for the effects of portal embolization. Digital analysis of immunohistochemistry targeting IBMIR mediators was performed at 1 and 24 h after intraportal islet infusion. Early findings observed in transplanted islets include complement and antibody deposition, and infiltration by neutrophils, macrophages and platelets. Insulin, complement, antibody, neutrophils, macrophages and platelets were similar between GTKO and WT islets, with increasing macrophage infiltration at 24 h in both phenotypes. This model provides an objective and internally controlled study of distinct islet preparations and documents the temporal histology of IBMIR.     

Adult porcine islets produce MCP-1 and recruit human monocytes in vitro

Abstract:  Type 1 diabetes can be cured by transplantation of isolated pancreatic islets. Because of the shortage of human donor tissue, adult porcine islets (APIs) constitute a possible alternative tissue source. Upon intraportal injection, islets are subjected to an instant blood-mediated inflammatory reaction (IBMIR) leading to blood clotting, leukocyte islet-infiltration, islet damage and insulin release. Xenogeneic islets surviving IBMIR are rejected in a cellular process involving CD4⁺ T lymphocytes and macrophages. We have investigated whether APIs themselves produce and secrete chemokines and/or inflammatory cytokines that may contribute to IBMIR and/or cell-mediated rejection. APIs, cultured for 1, 4, 8 and 11 days post-isolation, expressed mRNA for monocyte chemoattractant protein-1 (MCP-1), IL-1β and TNF-α. API culture supernatants induced migration of human monocytes, which was significantly blocked by an anti-human MCP-1 antibody (Ab). Immunohistochemistry revealed MCP-1 in the cytoplasm of α- and β-cells in isolated islets and in islets in situ. However, APIs or their supernatants were not able to activate human aortic endothelial cells (HAECs) in vitro, and neither IL-1β nor TNF-α were detected by enzyme-linked immunosorbent assay (ELISA) in API culture supernatants. Both recombinant porcine IL-1β and TNF-α were able to activate human endothelial cells (ECs) inducing CD62E and CD106 expression as analyzed by flow cytometry. In conclusion, MCP-1 secreted by APIs may contribute to both IBMIR and rejection by attracting monocytes into the islet; monocytes which upon transformation into macrophages will potentiate antigen presentation and execute islet rejection.     

Interference with tissue factor prolongs intrahepatic islet allograft survival in a nonhuman primate marginal mass model

BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.