Instant Blood-mediated Inflammatory Reaction Research Articles

The effect of hypoxia on free and encapsulated adult porcine islets-an in vitro study.

Adult porcine islets (APIs) constitute a promising alternative to human islets in treating type 1 diabetes. The intrahepatic site has been used in preclinical primate studies of API xenografts; however, an estimated two-thirds of donor islets are destroyed after intraportal infusion due to a number of factors, including the instant blood-mediated inflammatory reaction (IBMIR), immunosuppressant toxicity, and poor reestablishment of extracellular matrix connections. Intraperitoneal (ip) transplantation of non-vascularized encapsulated islets offers several advantages over intrahepatic transplantation of free islets, including avoidance of IBMIR, immunoprotection, accommodation of a larger graft volume, and reduced risk of hemorrhage. However, there exists evidence that the peritoneal site is hypoxic, which likely impedes islet function. We tested the effect of hypoxia (2%-5% oxygen or pO2 : 15.2-38.0mmHg) on free and encapsulated APIs over a period of 6days in culture. Free and encapsulated APIs under normoxia served as controls. Islet viability was evaluated with a viability/cytotoxicity assay using calcein AM and ethidium bromide on days 1, 3, and 6 of culture. Alamar blue assay was used to measure the metabolic activity on days 1 and 6. Insulin in spent medium was assayed by ELISA on days 1 and 6. Viability staining indicated that free islet clusters lost their integrity and underwent severe necrosis under hypoxia; encapsulated islets remained intact, even when they began to undergo necrosis. Under hypoxia, the metabolic activity and insulin secretion (normalized to metabolic activity) of both free and encapsulated islets decreased relative to islets cultured under normoxic conditions. Hypoxia (2%-5% oxygen or pO2 : 15.2-38.0mmHg) affects the viability, metabolic activity, and insulin secretion of both free and encapsulated APIs over a six-day culture period. Encapsulation augments islet integrity under hypoxia, but it does not prevent loss of viability, metabolic activity, or insulin secretion.

Genetic strategies to bring islet xenotransplantation to the clinic.

Despite the benefits of islet and pancreas allotransplantation, their widespread use in type 1 diabetes is limited because of the paucity of suitable donors. Porcine xenotransplantation offers an alternative, and advances in genetic modification of pigs have opened up new potential for its clinical use. This review outlines the barriers to successful islet xenotransplantation, and genetic modifications that have been tested to overcome these. Islets from pigs lacking α1,3-galactosyltransferase, to prevent hyperacute rejection, are now used as a background strain for further genetic modifications. The instant blood-mediated inflammatory reaction is overcome by expressing complement regulators including CD46, CD55 and CD59. Prevention of immune-mediated rejection mediated by T cells, macrophages and natural killer cells remains a challenge. The use of immunosuppressive antibodies, such as anti-CD154 or anti-CD2, can be protective, and may be useful if they are produced by the islets themselves. The field of xenotransplantation has benefited enormously from the development of new genetic modification strategies. With the possibility of multiple genetic modifications in the same animal, and a detailed knowledge of the mechanism of xenograft rejection, the challenge now is to develop islets that provide long-term graft survival without systemic immunosuppression.

Instant Blood-Mediated Inflammatory Reaction in Hepatocyte Transplantation: Current Status and Future Perspectives.

Hepatocyte transplantation (HT) is emerging as a promising alternative to orthotopic liver transplantation (OLT) in patients with certain liver-based metabolic disease and acute liver failure. Hepatocytes are generally infused into the portal venous system, from which they migrate into the liver cell plates of the native organ. One of the major hurdles to the sustained success of this therapy is early cell loss, with up to 70% of hepatocytes lost immediately following infusion. This is largely thought to be due to the instant blood-mediated inflammatory reaction (IBMIR), resulting in the activation of complement and coagulation pathways. Transplanted hepatocytes produce and release tissue factor (TF), which activates the coagulation pathway, leading to the formation of thrombin and fibrin clots. Thrombin can further activate a number of complement proteins, leading to the activation of the membrane attack complex (MAC) and subsequent hepatocyte cell death. Inflammatory cells including granulocytes, monocytes, Kupffer cells, and natural killer (NK) cells have been shown to cluster around transplanted hepatocytes, leading to their rapid clearance shortly after transplantation. Current research aims to improve cell engraftment and prevent early cell loss. This has been proven successful in vitro using pharmacological interventions such as melagatran, low-molecular-weight dextran sulphate, and N-acetylcysteine (NAC). Effective inhibition of IBMIR would significantly improve hepatocyte engraftment, proliferation, and function, providing successful treatment for patients with liver-based metabolic diseases.

Characterizing the Mechanistic Pathways of the Instant Blood-Mediated Inflammatory Reaction in Xenogeneic Neonatal Islet Cell Transplantation.

The instant blood-mediated inflammatory reaction (IBMIR) causes major loss of islets after transplantation and consequently represents the initial barrier to survival of porcine neonatal islet cell clusters (NICC) after xenotransplantation. This study used novel assays designed to characterize the various immunologic components responsible for xenogeneic IBMIR to identify initiators and investigate processes of IBMIR-associated coagulation, complement activation and neutrophil infiltration. The IBMIR was induced in vitro by exposing NICC to platelet-poor or platelet-rich human plasma or isolated neutrophils. We found that xenogeneic IBMIR was characterized by rapid, platelet-independent thrombin generation, with addition of platelets both accelerating and exacerbating this response. Platelet-independent complement activation was observed as early as 30 minutes after NICC exposure to plasma. However, membrane attack complex formation was not observed in NICC histopathology sections until after 60 minutes. We demonstrated for the first time that NICC-mediated complement activation was necessary for neutrophil activation in the xenogeneic IBMIR setting. Finally, using the Seahorse extracellular flux analyzer, we identified substantial loss of islet function (up to 40%) after IBMIR with surviving NICC showing evidence of mitochondrial damage. This study used novel assays to describe multiple key pathways by which xenogeneic IBMIR causes islet destruction, allowing further refinement of future interventions aimed at resolving the issue of IBMIR in xenotransplantation.

Characterization of Innate Immunity in an Extended Whole Blood Model of Human Islet Allotransplantation.

The instant blood-mediated inflammatory reaction (IBMIR) has been studied in whole blood models of human allo-islet transplantation for short periods (<6 h). Beyond this time frame the innate response to intraportally transplanted islets is less well described. A novel whole blood model was applied to study blood-islet-graft interactions up to 48 h. Heparinized polyvinyl chloride tubing was sealed into small bags containing venous blood together with allogeneic human islets and exocrine tissue, respectively. The bags were attached to a rotating wheel (37°C). Concentrated glucose and sodium hydrogen carbonate were added every 12 h to maintain physiological limits for sustained immune cell functions. Plasma was collected at repeated time points for analyses of coagulation/complement activation and chemokine/cytokine production. Immune cell infiltration was analyzed using immunohistochemistry. Coagulation and platelet activation markers, thrombin-antithrombin complex (TAT) and soluble CD40 ligand (sCD40L) showed early high concentrations (at 6-12 h). sC5b-9 steadily increased over 48 h. At 6 h neutrophils and monocytes surrounded the clotted cellular grafts with a following massive infiltration of neutrophils. High and increasing concentrations of CXCR1/2 ligands [IL-8 and growth-regulated oncogene α/β/γ (Gro-α/β/γ)] and IL-6 were produced in response to human islets and exocrine tissue. The CCR2 ligand monocyte chemoattractant protein 1 (MCP-1) exhibited increasing concentrations in response to exocrine tissue. The CXCR3 ligand interferon-inducible T cell α chemoattractant (I-TAC) was produced in response to both human islets and exocrine tissue from 6 h. Monokine induced by γ interferon (Mig) and interferon γ-induced protein 10 (IP-10) showed a later response, preferentially to exocrine tissue and with larger variations among preparations. An extended blood model of clinical islet transplantation allowed characterization of early immune activation in response to human islets and exocrine tissue. Increased production of chemokines targeting CXCR1/2, CCR2, and CXCR3 was observed, accompanied by massive intraislet neutrophil infiltration over 48 h. The model proved to be useful in exploring early blood-mediated reactions to cellular transplants and has relevance for evaluation of pharmacological interventions to prevent graft loss.

Initial in vitro studies on tissues and cells from GTKO/CD46/NeuGcKO pigs.

The impact that the absence of expression of NeuGc in pigs might have on pig organ or cell transplantation in humans has been studied in vitro, but only using red blood cells (pRBCs) and peripheral blood mononuclear cells (pPBMCs) as the target cells for immune assays. We have extended this work in various in vitro models and now report our initial results. The models we have used involve GTKO/hCD46 and GTKO/hCD46/NeuGcKO pig aortas and corneas, and pRBCs, pPBMCs, aortic endothelial cells (pAECs), corneal endothelial cells (pCECs), and isolated pancreatic islets. We have investigated the effect of the absence of NeuGc expression on (i) human IgM and IgG binding, (ii) the T-cell proliferative response, (iii) human platelet aggregation, and (iv) in an in vitro assay of the instant blood-mediated inflammatory reaction (IBMIR) following exposure of pig islets to human blood/serum. The lack of expression of NeuGc on some pig tissues (aortas, corneas) and cells (RBCs, PBMCs, AECs) significantly reduces the extent of human antibody binding. In contrast, the absence of NeuGc expression on some pig tissues (CECs, isolated islet cells) does not reduce human antibody binding, possibly due to their relatively low NeuGc expression level. The strength of the human T-cell proliferative response may also be marginally reduced, but is already weak to GTKO/hCD46 pAECs and islet cells. We also demonstrate that the absence of NeuGc expression on GTKO/hCD46 pAECs does not reduce human platelet aggregation, and nor does it significantly modify the IBMIR to pig islets. The absence of NeuGc on some solid organs from GTKO/hCD46/NeuGcKO pigs should reduce the human antibody response after clinical transplantation when compared to GTKO/hCD46 pig organs. However, the clinical benefit of using certain tissue (e.g., cornea, islets) from GTKO/hCD46/NeuGcKO pigs is questionable.

Developmental endothelial locus-1 modulates platelet-monocyte interactions and instant blood-mediated inflammatory reaction in islet transplantation.

Platelet-monocyte interactions are strongly implicated in thrombo-inflammatory injury by actively contributing to intravascular inflammation, leukocyte recruitment to inflamed sites, and the amplification of the procoagulant response. Instant blood-mediated inflammatory reaction (IBMIR) represents thrombo-inflammatory injury elicited upon pancreatic islet transplantation (islet-Tx), thereby dramatically affecting transplant survival and function. Developmental endothelial locus-1 (Del-1) is a functionally versatile endothelial cell-derived homeostatic factor with anti-inflammatory properties, but its potential role in IBMIR has not been previously addressed. Here, we establish Del-1 as a novel inhibitor of IBMIR using a whole blood-islet model and a syngeneic murine transplantation model. Indeed, Del-1 pre-treatment of blood before addition of islets diminished coagulation activation and islet damage as assessed by C-peptide release. Consistently, intraportal islet-Tx in transgenic mice with endothelial cell-specific overexpression of Del-1 resulted in a marked decrease of monocytes and platelet-monocyte aggregates in the transplanted tissues, relative to those in wild-type recipients. Mechanistically, Del-1 decreased platelet-monocyte aggregate formation, by specifically blocking the interaction between monocyte Mac-1-integrin and platelet GPIb. Our findings reveal a hitherto unknown role of Del-1 in the regulation of platelet-monocyte interplay and the subsequent heterotypic aggregate formation in the context of IBMIR. Therefore, Del-1 may represent a novel approach to prevent or mitigate the adverse reactions mediated through thrombo-inflammatory pathways in islet-Tx and perhaps other inflammatory disorders involving platelet-leukocyte aggregate formation.

A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers.

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.

Unexpected Massive Hemothorax After Pancreatic Islet Transplantation: A Case Report

The percutaneous transhepatic portal approach is the most commonly used technique for islet transplantation, largely owing to its safety and minimally invasive characteristic. Bleeding complications after islet transplantation are rare and include portal vein thrombosis and subcapsular liver hematoma. We report a massive hemothorax after portal vein catheterization in a patient with brittle type 1 diabetes undergoing hepatic islet embolization. The patient was under long-term aspirin therapy because of vascular complications and received heparin in low doses to prevent the instant blood-mediated inflammatory reaction and reduce the risk of portal vein thrombosis. The present case illustrates the particular risk of bleeding complications in patients with brittle type 1 diabetes, who represent a frail population. This uncommon adverse event highlights the importance of close monitoring in the first days following islet transplantation.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess β-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet β-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

In vitro exposure of pig neonatal isletlike cell clusters to human blood.

Pig islet grafts have been successful in treating diabetes in animal models. One remaining question is whether neonatal pig isletlike cell clusters (NICC) are resistant to the early loss of islets from the instant blood-mediated inflammatory reaction (IBMIR). Neonatal isletlike cell clusters were harvested from three groups of piglets-(i) wild-type (genetically unmodified), (ii) α1,3-galactosyltransferase gene-knockout (GTKO)/CD46, and (iii) GTKO/CD46/CD39. NICC samples were mixed with human blood in vitro, and the following measurements were made-antibody binding; complement activation; speed of islet-induced coagulation; C-peptide; glutamic acid decarboxylase (GAD65) release; viability. Time to coagulation and viability were both reduced in all groups compared to freshly drawn non-anticoagulated human blood and autologous combinations, respectively. Antibody binding to the NICC occurred in all groups. Neonatal isletlike cell clusters were subject to humoral injury with no difference associated to their genetic characteristics.

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.

Engineering more efficient multipotent mesenchymal stromal (stem) cells for systemic delivery as cellular therapy

Multipotent mesenchymal stromal (stem) cells (MSCs) harbour great potential for regenerative and immunomodulatory therapies. A major drawback in MSC therapy appears to be the incomplete understanding of cell fate and function following systemic administration. We here summarize our knowledge on MSC interaction with human blood. Analysis of tissues following MSC therapy indicated limited long‐term engraftment. MSCs and other stromal cells trigger an innate immune attack, termed instant blood‐mediated inflammatory reaction (IBMIR), which has been shown to compromise the survival, engraftment and function of cellular therapeutics. MSCs express hemostatic regulators similar to those produced by endothelial cells, but display higher amounts of prothrombotic tissue factor on their surface, which triggers the IBMIR after blood exposure. This process is dependent on the cell dose, the choice of MSC donor, the cell passage number and cell handling before infusion. Freshly harvested, short‐term expanded bone marrow MSCs trigger only weak blood responses, while extended culture, freeze‐thawing, washing and resupsension in human AB plasma, and co‐culture with activated lymphocytes increases their prothrombotic profile. After MSC infusion to patients, we found increased formation of blood activation markers, but no formation of hyperfibrinolysis marker D‐dimer or acute phase reactants with the currently applied dose of 1–3 × 106 cells/kg, demonstrating product safety. We conclude, that currently applied doses of low‐passage clinical grade MSCs are safe and elicit only minor systemic effects, but higher cell doses, and particularly higher passage cells, should be handled with care. This deleterious reaction can compromise the survival, engraftment and function of these therapeutic cells.

Regulation of Instant Blood Mediated Inflammatory Reaction (IBMIR) in Pancreatic Islet Xeno-Transplantation: Points for Therapeutic Interventions.

Xeno-transplantation of pancreatic islets represents a promising therapeutic alternative for the treatment of type 1 diabetes mellitus. However, potent innate immune responses induced shortly after the transplantation of donor islets to the recipient, comprising the Instant Blood Mediated Immune Reaction (IBMIR), exert detrimental actions on islet graft function. The coagulation and complement cascades together with the leukocyte and platelet populations are the major players in IBMIR. This innate immune attack affects dramatically islet integrity and leads to significant loss of function of the xenograft. In the present review, we focus on the mechanisms contributing to IBMIR components and address therapeutic intervention approaches to limit IBMIR by administering inhibitors in circulation, by coating the islet surface with inhibitors or by generating transgenic donor animals; these approaches could result in improved xenograft survival.

Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties?

We have recently reported that therapeutic mesenchymal stromal cells (MSCs) have low engraftment and trigger the instant blood mediated inflammatory reaction (IBMIR) after systemic delivery to patients, resulting in compromised cell function. In order to optimize the product, we compared the immunomodulatory, blood regulatory, and therapeutic properties of freeze-thawed and freshly harvested cells. We found that freeze-thawed MSCs, as opposed to cells harvested from continuous cultures, have impaired immunomodulatory and blood regulatory properties. Freeze-thawed MSCs demonstrated reduced responsiveness to proinflammatory stimuli, an impaired production of anti-inflammatory mediators, increased triggering of the IBMIR, and a strong activation of the complement cascade compared to fresh cells. This resulted in twice the efficiency in lysis of thawed MSCs after 1 hour of serum exposure. We found a 50% and 80% reduction in viable cells with freshly detached as opposed to thawed in vitro cells, indicating a small benefit for fresh cells. In evaluation of clinical response, we report a trend that fresh cells, and cells of low passage, demonstrate improved clinical outcome. Patients treated with freshly harvested cells in low passage had a 100% response rate, twice the response rate of 50% observed in a comparable group of patients treated with freeze-thawed cells at higher passage. We conclude that cryobanked MSCs have reduced immunomodulatory and blood regulatory properties directly after thawing, resulting in faster complement-mediated elimination after blood exposure. These changes seem to be paired by differences in therapeutic efficacy in treatment of immune ailments after hematopoietic stem cell transplantation.

Liver DX5- Natural Killer Cells Play a Crucial Role On Instant Blood-Mediated Inflammatory Reaction After Islet Transplantation.

Introduction: Destruction of islet grafts by the instant blood-mediated inflammatory reaction (IBMIR) and the immune response after islet transplantation remain a major obstacle in islet transplantation. We have previously shown that natural killer (NK) cells in the liver play a key role in early inflammatory events that adversely affect islet function and engraftment in the liver. Here, we investigated the relationship between the IBMIR and activation of liver NK cells after islet transplantation by analyzing phenotypic alterations in liver NK cells under proinflammatory stress-mimicking IBMIR conditions in vitro. Methods: Liver NK cells from C57BL6 mice were cultured for 24 h in the presence of IFNγ, TNFα and IL-1β, which were predominantly and transiently produced on the IBMIR conditions. The expression of activation marker CD69 and TNF-related apoptosis-inducing ligand (TRAIL) on liver NK cells was analyzed by flow cytometry. Results: Liver NK cells were classified into subsets of DX5-NK1.1+ or conventional DX5+ NK1.1+ NK cells. Liver NK cells contained approximately seven times as many DX5- NK cells as splenic NK cells. In naive conditions, the proportions of CD69+ and TRAIL+ NK cells in the DX5- NK cell were significantly higher than in DX5+ NK cells (CD69: 84.1 ± 7.5% vs 12.8 ± 4.2%, TRAIL: 70.8 ± 9.3% vs 4.9 ± 2.2%, respectively), suggesting that DX5- NK cells are in an activation stage even without any stimulation. The expression of these markers on splenic NK cells was similar to that on liver DX5+ NK cells. The proportion of CD69+ NK cells was not increased when cultured with IFNγ, TNFα, or IL-1β alone (24h/fresh ratio; IFNγ 0.79, p=0.29; TNFα 1.24, p=0.31; IL-1β 0.63, p=0.14). However, the proportion of CD69+ NK cells was significantly increased only when all three cytokines were simultaneously added (24h/fresh ratio; 2.00, p<0.01). Furthermore, the population of DX5- CD69+ NK cells expanded under co-culture with all three cytokines (27.5%±5.8% vs. 65.7±13.8%, p<0.01), whereas DX5+ CD69+ NK cells did not. In addition, IFNγ production was promoted in DX5- NK cells. Conclusion: Liver-resident DX5-NK cells play a crucial role in the liver NK cells, and activation of these NK cells was promptly induced by IBMIR circumstance. Thus, targeting the elimination or inactivation of liver-resident DX5- NK cells may improve the engraftment of transplanted islets without inhibiting the innate immune system.

Coatting NICCs With MSCs and EPCs to Alleviate Instant Blood-Mediated Inflammatory Reaction.

Background and aim.Transplantation of islets of porcine is a promising technique for treating insulin-dependent diabetes mellitus (type I). Instant blood-mediated inflammatory reaction (IBMIR) causes rapid islet loss in portal vein islet transplantation. Since it has been all known endothelial progenitor cells (EPCs) can aid neovascularization and protect against complement mediated lysis and activation of coagulation. Mesenchymal stem cells (MSCs) can enhace the viability of NICCs and modulate immune response. Thus we'd like to know when transplantation of the NICC coated with EPCs and MSCs would aggravate the IBMIR or alleviate it ? Research Design and methods. Porcine islets were coated with P2-P4 human umbilicl cord derived MSCs and human cord blood derived EPCs in specially modified culture medium composed of EGM2, porcine serum, IBMX and nicotinamide for 5 days. The capacity of EPCs and MSCs adhere to and grow into porcine islets was analyzed by fluorescence microscopy and confocal scanning microscopy with different cell tracers. The survival and functionality of these composite islets were evaluated by AO/EB and static assay. A negative control group, NICC alone group, and the composite islets group were examined with an in vitro tubing loop assay using human blood. Platelet consumption and complement and coagulation activation were assessed by platelet count, C3a, and thrombin-antithrombin complex (TAT), respectively. Results. MSCs can improve the EPCs coverage compared with EPC islets alone. From the first two days the coverage area was increased rapidly and the majority of NICC can be covered more than 80%. Then they were almost no change at all. Islet survival in vitro and the functionality of the composite islets after culture were almost the same to those of control islets. After incubation with human blood, the composite islets group showed platelet consumption inhibition and low C3a and TAT assay results compared to uncoated controls. Conclusions. Compared to the uncoated controls, the MSCs and EPCs coated NICC could alleviate the IBMIR. And this may because the MSCs can help the EPCs covering the NICC then reduce the exposure of NICC to the human blood. These findings might provide directly applicable tool to enhance the efficacy of islet transplantation in clinical practice.

The Instant Blood Mediated Inflammatory Reaction in a Dual Transplant Model of Islet Transplantation.

The Instant Blood Mediated Inflammatory Reaction in a Dual Transplant Model of Islet Transplantation.

Ultrasound Molecular Detection of Instant Blood-Mediated Inflammatory Reaction Induced By Pig Islet Xenotransplantation.

Ultrasound Molecular Detection of Instant Blood-Mediated Inflammatory Reaction Induced By Pig Islet Xenotransplantation.

Control of IBMIR in Neonatal Porcine Islet Xenotransplantation in Baboons

The instant blood-mediated inflammatory reaction (IBMIR) is a major obstacle to the engraftment of intraportal pig islet xenografts in primates. Higher expression of the galactose-α1,3-galactose (αGal) xenoantigen on neonatal islet cell clusters (NICC) than on adult pig islets may provoke a stronger reaction, but this has not been tested in the baboon model. Here, we report that WT pig NICC xenografts triggered profound IBMIR in baboons, with intravascular clotting and graft destruction occurring within hours, which was not prevented by anti-thrombin treatment. In contrast, IBMIR was minimal when recipients were immunosuppressed with a clinically relevant protocol and transplanted with NICC from αGal-deficient pigs transgenic for the human complement regulators CD55 and CD59. These genetically modified (GM) NICC were less susceptible to humoral injury in vitro than WT NICC, inducing significantly less complement activation and thrombin generation when incubated with baboon platelet-poor plasma. Recipients of GM NICC developed a variable anti-pig antibody response, and examination of the grafts 1 month after transplant revealed significant cell-mediated rejection, although scattered insulin-positive cells were still present. Our results indicate that IBMIR can be attenuated in this model, but long-term graft survival may require more effective immunosuppression or further donor genetic modification.