Low molecular weight dextran sulfate prevents the instant blood-mediated inflammatory reaction induced by adult porcine islets

BACKGROUND: One of the main obstacles to clinical islet xenotransplantation is the injurious instant blood-mediated inflammatory reaction (IBMIR) that causes rapid binding of platelets to the islet surface, activation of the coagulation and complement systems, and leukocyte infiltration of the islets when the islets are exposed to blood. METHODS: This study assesses the effect of low molecular weight dextran sulfate (LMW-DS) on IBMIR induced by porcine islets in an in vitro tubing loop assay using human blood and in an in vivo model using diabetic athymic mice. RESULTS: In vitro experiments demonstrated that platelet consumption, coagulation, and complement activation were already reduced in the presence of LMW-DS at 0.01 mg/mL, and that at 0.1 mg/mL, LMW-DS prevented IBMIR. Immunohistochemical investigation showed that the leukocyte infiltration was abrogated at the highest dose. In vivo experiments showed that the transplanted pig islets survived for a significantly longer period in recipients treated with LMW-DS, and morphologic examination of transplanted islets showed a reduction in IBMIR analogous to that demonstrated by in vitro studies. CONCLUSIONS: Given that LMW-DS has been used in clinical studies without serious adverse reactions, it has potential as a drug candidate that can control the strong innate immune response induced by pig islets when transplanted through the portal vein.     

Low-molecular weight dextran sulfate abrogates the instant blood-mediated inflammatory reaction induced by adult porcine islets both in vitro and in vivo

BACKGROUND: One of the main obstacles to clinical application of islet xenotransplantation is the injurious inflammatory reaction elicited by porcine islets when they are exposed to fresh human blood in vitro and in vivo. This instant blood-mediated inflammatory reaction (IBMIR) causes rapid binding of platelets to the islet surface, activation of the coagulation and complement systems, and leukocyte infiltration of the islets. As a consequence of IBMIR, morphological destruction of porcine islets occurs within the first few hours after transplantation. MATERIALS AND METHODS: In the present study, by analyzing the plasma samples and performing immunohistochemical investigation, we assessed the effect of adding low-molecular weight dextran sulfate (LMW-DS) at 0.01-1 mg/mL to an in vitro tubing loop assay in which porcine islets were exposed to fresh human blood. The effect of LMW-DS also was investigated in an in vivo model using diabetic athymic mice, which provides an innate inflammatory milieu without influence of T cells. The possible toxicity of LMW-DS was assessed by culturing pig islets in the presence or absence of LMW-DS for 3 days. RESULTS: In the in vitro study, in the presence of LMW-DS at 0.01 mg/mL, platelet consumption, coagulation, and complement activation were reduced, and, at 0.1 mg/mL, LMW-DS totally prevented IBMIR. Immunohistochemical investigation showed that leukocyte infiltration was totally abrogated at the highest dose. A similar finding was observed in the in vivo study. No adverse effect of LMW-DS was observed on the quality of the islets. CONCLUSIONS: LMW-DS appears to be an effective drug candidate that is able to control the strong innate immune response induced by pig islets in contact with human blood.     

Dissecting the instant blood-mediated inflammatory reaction in islet xenotransplantation

Abstract: Background: A massive destruction of transplanted tissue occurs immediately following transplantation of pancreatic islets from pig to non‐human primates. The detrimental instant blood‐mediated inflammatory reaction (IBMIR), triggered by the porcine islets, is a likely explanation for this tissue loss. This reaction may also be responsible for mediating an adaptive immune response in the recipient that requires a heavy immunosuppressive regimen. Materials and methods: Low molecular weight dextran sulfate (LMW‐DS) and the complement inhibitor Compstatin were used in a combination of in vitro and in vivo studies designed to dissect the xenogeneic IBMIR in a non‐human primate model of pancreatic islet transplantation. Adult porcine islets (10 000 IEQs/kg) were transplanted intraportally into three pairs of cynomolgus monkeys that had been treated with LMW‐DS or heparin (control), and the effects on the IBMIR were characterized. Porcine islets were also incubated in human blood plasma in vitro to assess complement inhibition by LMW‐DS and Compstatin. Results: Morphological scoring and immunohistochemical staining revealed that the severe islet destruction and macrophage, neutrophilic granulocyte, and T‐cell infiltration observed in the control (heparin‐treated) animals were abrogated in the LMW‐DS‐treated monkeys. Both coagulation and complement activation were significantly reduced in monkeys treated with LMW‐DS, but IgM and complement fragments were still found on the islet surface. This residual complement activation could be inhibited by Compstatin in vitro. Conclusions: The xenogeneic IBMIR in this non‐human primate model is characterized by an immediate binding of antibodies that triggers deleterious complement activation and a subsequent clotting reaction that leads to further complement activation. The effectiveness of LMW‐DS (in vivo and in vitro) and Compstatin (in vitro) in inhibiting this IBMIR provides the basis for a protocol that can be used to abrogate the IBMIR in pig‐human clinical islet transplantation.     

Inhibition of thrombin abrogates the instant blood-mediated inflammatory reaction triggered by isolated human islets: possible application of the thrombin inhibitor melagatran in clinical islet transplantation

A thrombotic/inflammatory reaction is elicited when isolated islets of Langerhans come in contact with ABO-compatible blood. The detrimental effects of this instant blood-mediated inflammatory reaction (IBMIR) provide a reasonable explanation for the observation that an unexpectedly high number of islets, from several donors, are needed to produce normoglycemia in transplant patients with type 1 diabetes. In this study, the hypothesis that a specific thrombin inhibitor, Melagatran, could reduce IBMIR in an in vitro model in which human islets are exposed to ABO-compatible blood was tested. The administration of Melagatran abrogated IBMIR dose-dependently. Islets exposed to blood, in the absence or presence of 0.4 micromol/l Melagatran, exhibited a loss of integrity and were found to be trapped in macroscopic clots containing platelets and CD11b(+) leukocytes. At concentrations from 1 to 10 micromol/l, Melagatran inhibited both coagulation and complement activation. Also, platelet and leukocyte activation and consumption were decreased. Islet morphology was maintained with almost no platelets adhering to the surface, and infiltration by CD11b(+) leukocytes was considerably reduced. In conclusion, Melagatran significantly reduced IBMIR in this model system. This protective effect indicates that thrombin plays a pivotal role in IBMIR and suggests that thrombin inhibition can improve the outcome of clinical islet transplantation.     

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.     

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.     

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。     

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。     

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.     

Damage to porcine islets of Langerhans after exposure to human blood in vitro, or after intraportal transplantation to cynomologus monkeys: protective effects of sCR1 and heparin

BACKGROUND: Porcine islets offer an attractive alternative to human islets in clinical islet transplantation. The preferred method of islet transplantation is intra-portal injection into the liver. We have recently shown, both in vitro with human islets and in vivo with porcine islets, that islets exposed to allogeneic blood trigger an injurious inflammatory reaction characterized by activation of both coagulation and the complement systems. We have now tested whether a similar reaction is triggered when xenogeneic porcine islets are exposed to human blood in vitro and after intraportal transplantation into primates. Furthermore, we investigated the effect of inhibiting the complement and coagulation systems. METHOD: Islets isolated from adult and fetal porcine pancreas were perfused with fresh human blood in surface heparinized PVC tubings for 5-60 min. Blood cell counts and parameters related to coagulation and the complement system were analyzed, and islets were retrieved after the perifusion was examined by immunohistochemical method. Heparin and soluble complement receptor 1 (sCR1; TP10, 100 microg/ml) were added to the system in some experiments. Furthermore, adult porcine islets were transplanted intraportally into untreated and sCR1- (40 mg/kg BW i.v.) treated cynomolgus monkeys, and plasma insulin concentration was monitored during 60 min after transplantation. RESULTS: Porcine islets perifused with human blood triggered an immediate inflammatory reaction, characterized by a rapid consumption and activation of platelets, consumption of neutrophils and monocytes, activation of the coagulation and complement systems, and release of large amounts of insulin. Islet morphologic analysis revealed damaged islets embedded in clots and infiltrated with CD11+ leukocytes. C3a and C5b-9 was deposited on the islet surface, but human immunoglobulin was not. Complement inhibition with sCR1 reduced insulin release significantly. Intraportal islet transplantation into untreated cynomolgus monkeys resulted in a marked and rapid increase in plasma insulin concentration indicative of islet damage. Pretreatment of the monkeys with sCR1 resulted in significantly less insulin release than in untreated control monkeys. CONCLUSION: Exposure of isolated xenogeneic islets of Langerhans to blood, both in vitro and in vivo, resulted in acute islet damage. Complement and platelets seem to have a central role in the reactions described. Strategies to efficiently inhibit these reactions will be crucial for clinical intraportal islet xenotransplantation to be successful.     

Rapid loss of intraportally transplanted islets: an overview of pathophysiology and preventive strategies

Islets isolated from multiple pancreas donors are often necessary to achieve euglycemia in type 1 diabetic patients treated by islet allotransplantation. This increases the burden on the limited pool of donor organs. After infusion into the portal vein, a substantial percentage of islets are lost in the immediate post-transplant period through an inflammatory response termed the instant blood-mediated inflammatory reaction (IBMIR). IBMIR is equally, if not more of a problem after islet xenotransplantation, e.g., using pig islets in non-human primates. Coagulation, platelet aggregation, complement activation, and neutrophil and monocyte infiltration play roles in this reaction. IBMIR is potentially triggered by islet surface molecules, such as tissue factor and collagen residues that are normally not in direct contact with the blood. Also, stress during the islet isolation process results in the expression and production of several inflammatory mediators by the islets themselves. The potential mechanisms involved in this rapid graft loss and treatment options to reduce this loss are reviewed. Preventive strategies for IBMIR can include systemic treatment of the recipient, pre-conditioning of the isolated islets, or, in the case of xenotransplantation, genetic modification of the organ-source pig. Pre-conditioning of islets in culture by exposure to anti-inflammatory agents or by genetic modification harbors fewer risks of systemic complications in the recipient. The future of clinical islet transplantation will, at least in part, depend on the success of efforts made to reduce rapid graft loss, and thus allow islet transplantation to become a more efficient therapy by the use of single donors.     

Nicotinamide inhibits tissue factor expression in isolated human pancreatic islets: implications for clinical islet transplantation

BACKGROUND: Islet-produced tissue factor (TF) triggers an adverse clotting reaction, the instant blood-mediated inflammatory reaction (IBMIR), providing a likely explanation for the need of tissue from multiple donors in clinical islet transplantation. In this study, the authors investigated whether compounds previously shown to affect TF and macrophage chemoattractant protein (MCP)-1 expression in monocytes and endothelial cells have the same effect in human islet cells. METHODS: Islets were cultured in the presence of l-arginine, cyclosporine A, enalapril, or nicotinamide for 48 hr, after which the TF content and MCP-1 expression were assessed. The effect of nicotinamide on IBMIR was evaluated by exposing the treated islets to fresh human ABO-compatible blood in an in vitro loop model. RESULTS: Nicotinamide was the only compound that significantly reduced both TF and MCP-1. This reduction was dose-dependent. The level of MCP-1 was strongly correlated with TF expression (r2=0.98). In addition, the level of TF was also correlated with the ability of the islets to initiate IBMIR (r2=0.94). CONCLUSIONS: TF and MCP-1 expression in human islets can be decreased by adding nicotinamide to the culture medium. These observations indicate that the adverse effects of IBMIR in clinical islet transplantation could be reduced without endangering the recipient using antithrombotic drugs.     

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.     

Analysis of large-scale nonhuman primate islet isolations

AIMS: It is important to have clinically relevant large animal models, especially nonhuman primates, to improve the efficacy of islet isolation and transplantation prior to clinical trials. The aim of this study was to improve the efficacy of islet isolation by analyzing large-scale nonhuman primate islet isolations. METHODS: Sixty-one islet isolations were evaluated using nonhuman primates. An automated isolation method was scaled down for islet isolation. Islet yields of prepurification, postpurification, and postculture, purity of islets, viability of islets, and functionality with glucose stimulation test were assessed. Initially, we analyzed relationships between endpoints then analyzed additional factors for successful islet isolation. Those factors included donor characteristics, the two-layer method (TLM) of pancreas preservation, trypsin inhibition during digestion, and digestion and collection time. RESULTS: Prepurification islet yields were strongly correlated with postpurification yields and postculture yields. It weakly but significantly correlated with purity, viability, and functionality. The average prepurification yield was 16,267 IE/g with each case divided into either above-average (high-yield group) or below-average groups (low-yield group). In 8 cases, TLM and trypsin inhibition were used and all cases belonged to the high-yield group. There were no significant differences between high- and low-yield groups in terms of donor age, body weight, pancreas weight, and cold ischemic time. The high-yield group had significantly longer digestion times and shorter collection times. CONCLUSIONS: TLM, trypsin inhibition, complete digestion, and quick collections were key for successful islet isolation. Analysis of nonhuman primate islet isolation techniques provided useful information, which should help to improve clinical islet transplantation.     

Acute antibody-mediated complement activation mediates lysis of pancreatic islets cells and may cause tissue loss in clinical islet transplantation

BACKGROUND: Clinical islet transplantation is associated with loss of transplanted islets necessitating tissue from more than one donor to obtain insulin independence. The instant blood-mediated inflammatory reaction (IBMIR) is one explanation to the tissue loss. Complement activation is an important cytotoxic component of the IBMIR, and in the present study, we have investigated this component in detail. METHODS: Isolated human islets were analyzed by large particle flow cytometry and confocal microscopy after incubation in human ABO-compatible hirudin-plasma. RESULTS: After incubation in plasma, the islets bound IgG and IgM, CIq, C4, C3 and C9. The binding of C3b/iC3b was evident already after 5 min. The binding of C3b/iC3b and the generation of C3a and sC5b-9 were inhibited by the complement inhibitor Compstatin. Lysis as reflected by propidium iodide (PI) staining and release of C-peptide was also inhibited by Compstatin. There were significant correlations between IgM/IgG versus C3b/iC3b and between sC5b-9 and C-peptide. CONCLUSION: The conclusion is that complement is activated by natural IgG and IgM antibodies already after 5 min. The complement activation leads to lysis of cells of the pancreatic islets. This very rapid reaction may be an essential entity of the damage induced by the IBMIR in clinical islet transplantation.     

Tirofiban and Activated Protein C Synergistically Inhibit the Instant Blood Mediated Inflammatory Reaction (IBMIR) from Allogeneic Islet Cells Exposure to Human Blood

Instant blood mediated inflammatory reaction (IBMIR) occurs when islets are exposed to blood and manifests clinically as portal vein thrombosis and graft failure. The aim of this study was to determine the impact of recombinant human activated protein C (rhAPC) and platelet inhibition on IBMIR in order to develop a better targeted treatment for this condition. Five thousand human islet cell equivalents (IEQ) were mixed in a PVC loop system with 7 mL of ABO compatible human blood and incubated with rhAPC, either alone or in combination with tirofiban. Admixing human islets and blood caused rapid clot formation, consumption of platelets, leukocytes, fibrinogen, coagulation factors and raised d -dimers. Islets were encased in a fibrin and platelet clot heavily infiltrated with neutrophils. Tirofiban monotherapy was ineffective, whereas rhAPC monotherapy prevented IBMIR in a dose-dependent manner, preserving islet integrity while maintaining platelet and leukocyte counts, fibrinogen and coagulation factor levels, and reducing d -dimer formation. The combination of tirofiban and low-dose rhAPC inhibited IBMIR synergistically with an efficacy equal to high dose rhAPC. Tirofiban and rhAPC worked synergistically to preserve islets, suggesting that co-inhibition of the platelet and coagulation pathways' contribution to thrombin generation is required for the optimal anti-IBMIR effect.     

A simple and cost-effective method for the isolation of islets from nonhuman primates

Recent advances in islet cell transplantation have led to insulin independence in a majority of islet transplant recipients. However, there exists a need to overcome the shortage of donor tissue and the necessity for life-long immunosuppression. Preclinical studies in large animal models are necessary to evaluate the safety and efficacy of alternative approaches for clinical islet transplantation. The nonhuman primate serves as an appropriate animal model for such investigations; however, a major impediment in performing such preclinical research has been the difficulty in isolating islets of sufficient quantity and quality. The current study describes a simple and cost-effective method to isolate nonhuman primate islets to support preclinical islet transplantation research. The results of islet isolations from 54 cynomolgus monkeys and 4 baboons are reported. The pancreas was infused with Liberase HI and subjected to static digestion. The digested tissue was shaken, filtered through a mesh screen, applied to a discontinuous gradient, and centrifuged in much the same manner as with conventional rodent islet isolations. Islets were collected from the two interfaces, washed, and transplanted. Following purification, cynomolgus monkey islet isolation yields were 50,100 +/- 3120 IE total or 8760 +/- 420 IE/g pancreas with the percent purity and viability of 90.8 +/- 0.9 and 90.7 +/- 0.7, respectively. Total insulin content of the isolated islets was 405 +/- 53 microg insulin with DNA content being and 976 +/- 117 microg DNA, corresponding to a ratio of 0.57 microg insulin/microg DNA. STZ-induced diabetes was reversed in both mouse and nonhuman primate recipients, which possessed significant levels of c-peptide following transplantation and well-granulated islet grafts. The technique yields sufficient numbers of pure and viable islets to support preclinical research to develop improved strategies to prevent the immune destruction of the transplanted islet graft.     

Incompatibility between human blood and isolated islets of Langerhans: a finding with implications for clinical intraportal islet transplantation?

The remarkable difference in success rates between clinical pancreas transplantation and islet transplantation is poorly understood. Despite the same histocompatibility barrier and similar immunosuppressive treatments in both transplantation procedures, human intraportal islet transplantation has a much inferior success rate than does vascularized pancreas transplantation. Thus far, little attention has been directed to the possibility that islets transplanted into the blood stream may elicit an injurious incompatibility reaction. We have tested this hypothesis in vitro with human islets and in vivo with porcine islets. Human islets were exposed to nonanticoagulated human ABO-compatible blood in surface-heparinized polyvinyl chloride tubing loops. Heparin and/or the soluble complement receptor 1 (sCR1) TP10 were tested as additives. Adult porcine islets were transplanted intraportally into pigs, and the liver was recovered after 60 min for immunohistochemical staining. Human islets induced a rapid consumption and activation of platelets. Neutrophils and monocytes were also consumed, and the coagulation and complement systems were activated. Upon histological examination, islets were found to be embedded in clots and infiltrated with CD11+ leukocytes. Furthermore, the cellular morphology was disrupted. When heparin and sCR1 were added to the blood, these events were avoided. Porcine islets retrieved in liver biopsies after intraportal islet allotransplantation showed a morphology similar to that of human islets perifused in vitro. Thus, exposure of isolated islets of Langerhans to allogenic blood resulted in significant damage to the islets, a finding that could explain the unsatisfactory clinical results obtained with intraportal islet transplantation. Because administration of heparin in combination with a soluble complement receptor abrogated these events, such treatment would presumably improve the outcome of clinical islet transplantation by reducing both initial islet loss and subsequent specific immune responses.     

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.     

Discordant cellular and organ transplantation from bench to bedside

Allogeneic transplantation of human islets improves metabolic control in patients that suffer from type 1 diabetes but is limited by the supply of sufficient numbers of human donor islets. Therefore, the xenogenic transplantation of porcine islets potentially represents an attractive alternative. The immediate blood‐mediated inflammatory reaction (IBMIR) that results in early graft rejection is one of the major hurdles preventing xenotransplantation from clinical applications. Thus, characterizing and modulating IBMIR could be the key to realize clinical porcine islet transplantation. IBMIR is mediated by multiple components of the innate immune system and in order to assess composition and function of distinct myeloid cell types involved in the rejection process, we established a humanized mouse model to study IBMIR in vivo. We combined immune deficient mice that lack T, B, and NK cells with a mutant Kit receptor affecting the function of HSCs allowing for robust engraftment of HSCs and mature myeloid cells types over long periods of time. We could show that human myeloid cells repopulate peripheral organs such as spleen and liver – a prerequisite for the study of IBMIR in a humanized mouse model. We will now engage these mice to transplant porcine islets into the liver via the portal vein to induce immediate rejection of the graft. In this scenario presence of human myeloid cells in the liver of our new mouse model will facilitate a detailed characterization of the human IBMIR against pig islets and will also allow attempts to suppress this immune reaction by specific targeting of human leukocytes.