Instant Blood-mediated Inflammatory Reaction Research Articles

Engineering Pancreatic Islets to Transiently Codisplay on Their Surface Thrombomodulin and CD47 Immunomodulatory Proteins as a Means of Mitigating Instant Blood-Mediated Inflammatory Reaction following Intraportal Transplantation.

Most pancreatic islets are destroyed immediately after intraportal transplantation by an instant blood-mediated inflammatory reaction (IBMIR) generated through activation of coagulation, complement, and proinflammatory pathways. Thus, effective mitigation of IBMIR may be contingent on the combined use of agents targeting these pathways for modulation. CD47 and thrombomodulin (TM) are two molecules with distinct functions in regulating coagulation and proinflammatory responses. We previously reported that the islet surface can be modified with biotin for transient display of novel forms of these two molecules chimeric with streptavidin (SA), that is, thrombomodulin chimeric with SA (SA-TM) and CD47 chimeric with SA (SA-CD47), as single agents with improved engraftment following intraportal transplantation. This study aimed to test whether islets can be coengineered with SA-TM and SA-CD47 molecules as a combinatorial approach to improve engraftment by inhibiting IBMIR. Mouse islets were effectively coengineered with both molecules without a detectable negative impact on their viability and metabolic function. Coengineered islets were refractory to destruction by IBMIR ex vivo and showed enhanced engraftment and sustained function in a marginal mass syngeneic intraportal transplantation model. Improved engraftment correlated with a reduction in intragraft innate immune infiltrates, particularly neutrophils and M1 macrophages. Moreover, transcripts for various intragraft procoagulatory and proinflammatory agents, including tissue factor, HMGB1 (high-mobility group box-1), IL-1β, IL-6, TNF-α, IFN-γ, and MIP-1α, were significantly reduced in coengineered islets. These data demonstrate that the transient codisplay of SA-TM and SA-CD47 proteins on the islet surface is a facile and effective platform to modulate procoagulatory and inflammatory responses with implications for both autologous and allogeneic islet transplantation.

Inhibition of Toll-like Receptor 4 Using Small Molecule, TAK-242, Protects Islets from Innate Immune Responses.

Islet transplantation is a therapeutic option to replace β-cell mass lost during type 1 or type 3c diabetes. Innate immune responses, particularly the instant blood-mediated inflammatory reaction and activation of monocytes, play a major role in the loss of transplanted islet tissue. In this study, we aimed to investigate the inhibition of toll-like receptor 4 (TLR4) on innate inflammatory responses. We first demonstrate a significant loss of graft function shortly after transplant through the assessment of miR-375 and miR-200c in plasma as biomarkers. Using in vitro models, we investigate how targeting TLR4 mitigates islet damage and immune cell activation during the peritransplant period. The results of this study support the application of TAK-242 as a therapeutic agent to reduce inflammatory and innate immune responses to islets immediately following transplantation into the hepatic portal vein. Therefore, TLR4 may serve as a target to improve islet transplant outcomes in the future.

A brief review of the current status of pig islet xenotransplantation.

An estimated 1.5 million Americans suffer from Type I diabetes mellitus, and its incidence is increasing worldwide. Islet allotransplantation offers a treatment, but the availability of deceased human donor pancreases is limited. The transplantation of islets from gene-edited pigs, if successful, would resolve this problem. Pigs are now available in which the expression of the three known xenoantigens against which humans have natural (preformed) antibodies has been deleted, and in which several human 'protective' genes have been introduced. The transplantation of neonatal pig islets has some advantages over that of adult pig islets. Transplantation into the portal vein of the recipient results in loss of many islets from the instant blood-mediated inflammatory reaction (IBMIR) and so the search for an alternative site continues. The adaptive immune response can be largely suppressed by an immunosuppressive regimen based on blockade of the CD40/CD154 T cell co-stimulation pathway, whereas conventional therapy (e.g., based on tacrolimus) is less successful. We suggest that, despite the need for effective immunosuppressive therapy, the transplantation of 'free' islets will prove more successful than that of encapsulated islets. There are data to suggest that, in the absence of rejection, the function of pig islets, though less efficient than human islets, will be sufficient to maintain normoglycemia in diabetic recipients. Pig islets transplanted into immunosuppressed nonhuman primates have maintained normoglycemia for periods extending more than two years, illustrating the potential of this novel form of therapy.

Targeted mapping and utilization of the perihepatic surface for therapeutic beta cell replacement and retrieval in diabetic non-human primates

IntroductionSuccessful diabetes reversal using pancreatic islet transplantation by various groups illustrates the significant achievements made in cell-based diabetes therapy. While clinically, intraportal islet delivery is almost exclusively used, it is not without obstacles, including instant blood-mediated inflammatory reaction (IBMIR), relative hypoxia, and loss of function over time, therefore hindering long-term success. Here we demonstrate the perihepatic surface of non-human primates (NHPs) as a potential islet delivery site maximizing favorable characteristics, including proximity to a dense vascular network for adequate oxygenation while avoiding IBMIR exposure, maintenance of portal insulin delivery, and relative ease of accessibility through minimally invasive surgery or percutaneous means. In addition, we demonstrate a targeted mapping technique of the perihepatic surface, allowing for the testing of multiple experimental conditions, including a semi-synthetic hydrogel as a possible three-dimensional framework to improve islet viability.MethodsPerihepatic allo-islet cell transplants were performed in immunosuppressed cynomolgus macaques using a targeted mapping technique to test multiple conditions for biocompatibility. Transplant conditions included islets or carriers (including hydrogel, autologous plasma, and media) alone or in various combinations. Necropsy was performed at day 30, and histopathology was performed to assess biocompatibility, immune response, and islet viability. Subsequently, single-injection perihepatic allo-islet transplant was performed in immunosuppressed diabetic cynomolgus macaques. Metabolic assessments were measured frequently (i.e., blood glucose, insulin, C-peptide) until final graft retrieval for histopathology.ResultsTargeted mapping biocompatibility studies demonstrated mild inflammatory changes with islet-plasma constructs; however, significant inflammatory cell infiltration and fibrosis were seen surrounding sites with the hydrogel carrier affecting islet viability. In diabetic NHPs, perihepatic islet transplant using an autologous plasma carrier demonstrated prolonged function up to 6 months with improvements in blood glucose, exogenous insulin requirements, and HbA1c. Histopathology of these islets was associated with mild peri-islet mononuclear cell infiltration without evidence of rejection.DiscussionThe perihepatic surface serves as a viable site for islet cell transplantation demonstrating sustained islet function through 6 months. The targeted mapping approach allows for the testing of multiple conditions simultaneously to evaluate immune response to biomaterials at this site. Compared to traditional intraportal injection, the perihepatic site is a minimally invasive approach that allows the possibility for graft recovery and avoids IBMIR.

Neutrophil depletion for early allogeneic islet survival in a methacrylic acid (MAA) copolymer-induced, vascularized subcutaneous space

Islet transplantation is a promising treatment for type I diabetes (T1D). Despite the high loss of islets during transplantation, current islet transplant protocols continue to rely on portal vein infusion and intrahepatic engraftment. Because of the risk of portal vein thrombosis and the loss of islets to instant blood mediated inflammatory reaction (IBMIR), other transplantation sites like the subcutaneous space have been pursued for its large transplant volume, accessibility, and amenability for retrieval. To overcome the minimal vasculature of the subcutaneous space, prevascularization approaches or vascularizing biomaterials have been used to subcutaneously deliver islets into diabetic mice to return them to normoglycemia. Previous vascularization methods have relied on a 4 to 6 week prevascularization timeframe. Here we show that a vascularizing MAA-coated silicone tube can generate sufficient vasculature in 2 to 3 weeks to support a therapeutic dose of islets in mice. In order to fully harness the potential of this prevascularized site, we characterize the unique, subcutaneous immune response to allogeneic islets in the first 7 days following transplantation, a critical stage in successful engraftment. We identify neutrophils as a specific cellular target, a previously overlooked cell in the context of subcutaneous allogeneic islet transplantation. By perioperatively depleting neutrophils, we show that neutrophils are a key, innate immune cell target for successful early engraftment of allogeneic islets in a prevascularized subcutaneous site.

201.1: Co-engineering pancreatic islets with novel forms of thrombomodulin and CD47 as an effective means of mitigating instant blood-mediated inflammatory reaction

201.1: Co-engineering pancreatic islets with novel forms of thrombomodulin and CD47 as an effective means of mitigating instant blood-mediated inflammatory reaction

1496-P: Resatorvid, an Inhibitor of Toll-Like Receptor-4, Protects Allogenic Islets from Inflammatory and Immune Response

Purpose: Loss of islet mass by instant blood mediated inflammatory reaction (IBMIR) and immunological rejection are major hurdles currently facing the field of islet transplantation. Toll-like receptor 4 (TLR4) plays a central role in inflammation and shaping adaptive immune response. We investigated the protective effects of resatorvid (TAK-242) using in vitro models of islet transplantation. Methods: Soluble TAK-242 or TAK-242 conjugated to the surface of islets utilizing copper-free click chemistry with a cleavable chemical linker were used. Islets were mixed with allogenic blood for a period of 3 hours and plasma levels of miRNA-375 and miRNA-200c were analyzed by RT-qPCR. Allogenic peripheral blood mononuclear cells were cultured with islets. T cell and monocyte activation were monitored by flow cytometry. Cytokine and miRNA levels in culture supernatant were measured by Luminex assay and RT-qPCR respectively. Results: The presence of TAK-242 in soluble form as well as released by cleavable linker from islet surface significantly reduced the release of miRNA-375 (p<0.01) and miRNA-200c (p<0.01) indicating less islet damage. In allogenic co-culture of islets and PBMCs, TAK-242 inhibited the expression of CD69 in T cells as well as decreased (p<0.05) the expression of CD86 and CD80 in monocytes. The production of cytokines IL-1β (p<0.001), IL-6 (p=0.01), IL-8 (p<0.05), IP10 (p<0.05), MCP-1 (p<0.001) and TNFα (p=0.005) in culture supernatant was decreased in the presence of soluble TAK-242 as well as TAK-242 released from islet surfaces. Conclusions: Inhibition of TLR4 using small molecule, TAK-242, protected islets from innate damage due to IBMIR as well as adaptive responses of activation of T cells and monocytes. These results validate the future study of inhibition of TLR4 using in vivo transplant model(s) to further investigate the efficacy of TLR4 inhibition to improve islet transplant survival and function. Disclosure J.D.Mattke: None. B.Naziruddin: None. B.Kane: None.

Engineering islet surface with a novel form of thrombomodulin protein to mitigate instant blood-mediated inflammatory reaction following intraportal transplantation

Abstract Intraportal infusion of islets initiates an innate immune response termed instant blood-mediated inflammatory reaction (IBMIR) that is responsible for significant islet mass peritransplant. We herein used thrombomodulin (TM) as a multifaceted innate immune regulator to mitigate IBMIR to prevent the destruction of islets in a minimal mass syngeneic intraportal transplantation model. A novel form of TM molecule chimeric with streptavidin (SA-TM) was produced in insect cells and characterized for structure and attachment to biotinylated cell surface taking advantage of the high affinity interaction of biotin with streptavidin (SA). Islets were engineered with SA-TM and assessed for the ability to mitigate IBMIR in an in vitro blood loop assay and survival following intraportal transplantation (200 IEQ/mouse) in STZ diabetic mice (n=7). SA-engineered islets served as control (n=6). Animals were monitored for islet engraftment and subjected to various mechanistic studies. SA-TM prevented phagocytosis of xenogeneic cells by macrophages and inhibited neutrophil NETs formation. Engineering islets with SA-TM did not impact the viability and function of islets and prevented damage in a blood loop assay. SA-TM-engineered islets showed enhanced engraftment compared to SA-engineered islets (83% vs 29%) following transplantation. Enhanced engraftment was associated with decreased frequency of intragraft myeloid cells and expression of various inflammatory mediators, such as HMGB-1, tissue factor, and IL-1β. Engineering islets with the SA-TM protein provides a facile and clinically applicable approach to modulate innate immune responses for prevention of IBMIR with potential applications to allo/auto islet transplantation. Funded in parts by NIH U01AI132817 and R01AI121281 grants.

Research Highlights.

Research Highlights.

Engineering pancreatic islets with a novel form of thrombomodulin protein to overcome early graft loss triggered by instant blood-mediated inflammatory reaction

The instant blood-mediated inflammatory reaction (IBMIR) is initiated by innate immune responses that cause substantial islet loss after intraportal transplantation. Thrombomodulin (TM) is a multifaceted innate immune modulator. In this study, we report the generation of a chimeric form of thrombomodulin with streptavidin (SA-TM) for transient display on the surface of islets modified with biotin to mitigate IBMIR. SA-TM protein expressed in insect cells showed the expected structural and functional features. SA-TM converted protein C into activated protein C, blocked phagocytosis of xenogeneic cells by mouse macrophages and inhibited neutrophil activation. SA-TM was effectively displayed on the surface of biotinylated islets without a negative effect on their viability or function. Islets engineered with SA-TM showed improved engraftment and established euglycemia in 83% of diabetic recipients when compared with 29% of recipients transplanted with SA-engineered islets as control in a syngeneic minimal mass intraportal transplantation model. Enhanced engraftment and function of SA-TM–engineered islets were associated with the inhibition of intragraft proinflammatory innate cellular and soluble mediators of IBMIR, such as macrophages, neutrophils, high-mobility group box 1, tissue factor, macrophage chemoattractant protein-1, interleukin-1β, interleukin-6, tumor necrosis factor-α, interferon-γ. Transient display of SA-TM protein on the islet surface to modulate innate immune responses causing islet graft destruction has clinical potential for autologous and allogeneic islet transplantation.

Bioengineering the Vascularized Endocrine Pancreas: A Fine-Tuned Interplay Between Vascularization, Extracellular-Matrix-Based Scaffold Architecture, and Insulin-Producing Cells

Intrahepatic islet transplantation is a promising β-cell replacement strategy for the treatment of type 1 diabetes. Instant blood-mediated inflammatory reactions, acute inflammatory storm, and graft revascularization delay limit islet engraftment in the peri-transplant phase, hampering the success rate of the procedure. Growing evidence has demonstrated that islet engraftment efficiency may take advantage of several bioengineering approaches aimed to recreate both vascular and endocrine compartments either ex vivo or in vivo. To this end, endocrine pancreas bioengineering is an emerging field in β-cell replacement, which might provide endocrine cells with all the building blocks (vascularization, ECM composition, or micro/macro-architecture) useful for their successful engraftment and function in vivo. Studies on reshaping either the endocrine cellular composition or the islet microenvironment have been largely performed, focusing on a single building block element, without, however, grasping that their synergistic effect is indispensable for correct endocrine function. Herein, the review focuses on the minimum building blocks that an ideal vascularized endocrine scaffold should have to resemble the endocrine niche architecture, composition, and function to foster functional connections between the vascular and endocrine compartments. Additionally, this review highlights the possibility of designing bioengineered scaffolds integrating alternative endocrine sources to overcome donor organ shortages and the possibility of combining novel immune-preserving strategies for long-term graft function.

Dosing Limitation for Intra-Renal Arterial Infusion of Mesenchymal Stromal Cells.

The immunomodulatory and regenerative properties of mesenchymal stromal cells (MSCs) make MSC therapy a promising therapeutic strategy in kidney disease. A targeted MSC administration via the renal artery offers an efficient delivery method with limited spillover to other organs. Although local administration alleviates safety issues with MSCs in systemic circulation, it introduces new safety concerns in the kidneys. In a porcine model, we employed intra-renal arterial infusion of ten million allogenic adipose tissue-derived MSCs. In order to trigger any potential adverse events, a higher dose (hundred million MSCs) was also included. The kidney function was studied by magnetic resonance imaging after the MSC infusion and again at two weeks post-treatment. The kidneys were assessed by single kidney glomerular filtration rate (skGFR) measurements, histology and inflammation, and fibrosis-related gene expression. None of the measured parameters were affected immediately after the administration of ten million MSCs, but the administration of one hundred million MSCs induced severe adverse events. Renal perfusion was reduced immediately after MSC administration which coincided with the presence of microthrombi in the glomeruli and signs of an instant blood-mediated inflammatory reaction. At two weeks post-treatment, the kidneys that were treated with one hundred million MSCs showed reduced skGFR, signs of tissue inflammation, and glomerular and tubular damage. In conclusions, the intra-renal administration of ten million MSCs is well-tolerated by the porcine kidney. However, higher concentrations (one hundred million MSCs) caused severe kidney damage, implying that very high doses of intra-renally administered MSCs should be undertaken with caution.

Current Topics of Relevance to the Xenotransplantation of Free Pig Islets.

Pig islet xenotransplantation is a potential treatment for patients with type 1 diabetes. Current efforts are focused on identifying the optimal pig islet source and overcoming the immunological barrier. The optimal age of the pig donors remains controversial since both adult and neonatal pig islets have advantages. Isolation of adult islets using GMP grade collagenase has significantly improved the quantity and quality of adult islets, but neonatal islets can be isolated at a much lower cost. Certain culture media and coculture with mesenchymal stromal cells facilitate neonatal islet maturation and function. Genetic modification in pigs affords a promising strategy to prevent rejection. Deletion of expression of the three known carbohydrate xenoantigens (Gal, Neu5Gc, Sda) will certainly be beneficial in pig organ transplantation in humans, but this is not yet proven in islet transplantation, though the challenge of the ‘4th xenoantigen’ may prove problematic in nonhuman primate models. Blockade of the CD40/CD154 costimulation pathway leads to long-term islet graft survival (of up to 965 days). Anti-CD40mAbs have already been applied in phase II clinical trials of islet allotransplantation. Fc region-modified anti-CD154mAbs successfully prevent the thrombotic complications reported previously. In this review, we discuss (I) the optimal age of the islet-source pig, (ii) progress in genetic modification of pigs, (iii) the immunosuppressive regimen for pig islet xenotransplantation, and (iv) the reduction in the instant blood-mediated inflammatory reaction.

Effects of different routes of heparin on instant blood-mediated inflammatory reaction after portal vein islet transplantation.

Heparin is mainly used as an anticoagulant in clinic, and it also has a certain anti-inflammatory effect. At present, after portal vein islet transplantation in diabetic patients, heparin is mainly infused through the peripheral veins of the limbs to achieve the purpose of anticoagulation and protection of the graft, rather than through the portal vein. In this study, animal experiments were conducted to investigate the effect of heparin infusion via the portal vein and marginal ear vein on the instant blood-mediated inflammatory reaction (IBMIR) after portal vein islet transplantation, which is the choice of anticoagulation methods for clinical islet transplantation to provide a basis for decision-making. A total of 50 neonatal pigs (Xeno-1 type, 3-5 days) were selected. Islets were isolated and purified from the pancreas of neonatal pigs. Ten non-diabetic Landrace pigs (1.5-2.0 months) served as recipients, and 12 000 IEQ/kg neonatal porcine islets were transplanted into the liver through the portal vein. All recipients received bolus injection of 50 U/kg of heparin 10 minutes before transplantation. After the bolus injection of heparin, the experimental group received heparin via the portal vein [10 U/(kg·h), 5 recipients], and the control group received heparin via the marginal ear vein [10 U/(kg·h), 5 recipients]. The superior vena cava blood was collected from the 2 groups pre-operation at 1, 3, 24 h post-operation of the transplantation. The portal vein blood was collected from the experimental group at 1 and 3 h after the transplantation as well. The levels of complement C3a, C5a, thrombin-antithrombin complex (TAT), β-thromboglobulin (β-TG), and D-dimer as well as activated partial thromboplastin time (APTT) in superior vena cava blood from 1 and 3 h post-transplantation were detected in the 2 groups, and the levels of anti-Xa and anti-IIa in the portal vein and superior vena cava blood from 1 and 3 h post-transplantation in the experimental group were detected. Twenty four hours after the transplantation, the liver tissues in the 2 groups were collected for pathological examination to observe the inflammatory cell infiltration and peripheral thrombosis around the islets graft in liver. Before transplantation, there was no statistically significant difference in C3a, C5a, TAT, β-TG, D-dimer levels and APTT between the 2 groups (all P>0.05). At 1 and 3 h after transplantation, the C3a, TAT, and D-dimer levels in the experimental group were significant decreased than those in the control groups (all P<0.05), and at 3 h after transplantation the C5a was significant decreased than that in the control group (P<0.05). At 1 and 3 h after transplantation, the anti-Xa and anti-IIa levels in the portal vein blood were significantly increased than those in the superior vena cava blood in the experimental group (all P<0.05). Pathological results showed the presence of islet cell clusters in the liver blood vessels. The thrombus formation and neutrophil infiltration around islet graft was not obvious in the experimental group, while massive thrombus formation and neutrophil infiltration in the control group. Compared with marginal ear vein infusion of heparin, the direct infusion of heparin in the portal vein has a certain inhibitory effect on complement system, coagulation system activation and inflammatory cell infiltration in portal vein islet transplantation, which may attenuate the occurrence of IBMIR.

Finding Eden - alternative transplantation sites for pancreatic islets.

Pancreatic islets transplantation is an established treatment method for type 1 diabetic patients with the hypoglycemia unawareness syndrome in whom a therapy with modern technologies fails. Islet transplantation is most commonly done using an interventional radiology method: a tissue suspension of pancreatic islets is applied into a branch of the portal vein through a percutaneously installed catheter. Although being minimally invasive unlike pancreas organ transplant, this method is associated with many technical difficulties. Possible complications of the procedure include hemorrhage and portal vein thrombosis. Unlike their natural dispersed localization in exocrine pancreas, isolated pancreatic islets are exposed to hypoxia, toxins and immunosuppressive drugs in the liver parenchyma. Direct contact with the recipients blood causes an instant blood mediated inflammatory reaction (IBMIR) resulting in the death of more than half of the pancreatic islets shortly after their application. Therefore the size of the islet graft is often insufficient and a number of transplanted patients require administration of exogenous insulin. All of these are reasons for seeking an alternative transplantation site with more hospitable conditions for long-term islet survival. Various transplantation sites have been tested in experimental and clinical research. The advantages and disadvantages of some of them are summarized in this paper. Currently, transplantation into the greater omentum seems most promising, which has already been used in clinical practice at several institutions.

Bioluminescence Imaging In Vivo Confirms the Viability of Pancreatic Islets Transplanted into the Greater Omentum.

The liver is the most widely used site for pancreatic islet transplantation. However, several site-specific limitations impair functional success, with instant blood-mediated inflammatory reaction being the most important. The aim of this study was to develop a preclinical model for placement of the islet graft into a highly vascularized omental flap using a fibrin gel. For this purpose, we tested islet viability by bioluminescence imaging (BLI). Pancreatic islets were isolated from luciferase-positive and luciferase-negative rats, mixed at a 1:1 ratio, placed into a plasma-thrombin bioscaffold, and transplanted in standard (10 pancreatic islets/g wt; n = 10) and marginal (4 pancreatic islets/g wt; n = 7) numbers into the omentums of syngeneic diabetic animals. For the control, 4 pancreatic islets/g were transplanted into the liver using the standard procedure (n = 7). Graft viability was tested by bioluminescence at days 14, 30, 60, and 90 post transplant. Glucose levels, intravenous glucose tolerance, and serum C-peptide were assessed regularly. Nonfasting glucose levels < 10 mmol/l were restored in all animals. While islet viability in the omentum was clearly detected by stable luminescence signals throughout the whole study period, no signals were detected from islets transplanted into the liver. The bioluminescence signals were highly correlated with stimulated C-peptide levels detected at 80 days post transplant. Glucose tolerance did not differ among the 3 groups. We successfully tested a preclinical model of islet transplantation into the greater omentum using a biocompatible scaffold made from autologous plasma and human thrombin. Both standard and marginal pancreatic islet numbers in a gel-form bioscaffold placed in the omentum restored glucose homeostasis in recipients with diabetes. Bioluminescence was shown promising as a direct proof of islet viability.

Potential roles of mesenchymal stromal cells in islet allo- and xenotransplantation for type 1 diabetes mellitus.

Islet transplantation is poised to play an important role in the treatment of type 1 diabetes mellitus (T1DM). However, there are several challenges limiting its widespread use, including the instant blood-mediated inflammatory reaction, hypoxic/ischemic injury, and the immune response. Mesenchymal stem/stromal cells (MSCs) are known to exert regenerative, immunoregulatory, angiogenic, and metabolic properties. Here, we review recent reports on the application of MSCs in islet allo- and xenotransplantation. We also document the clinical trials that have been undertaken or are currently underway, relating to the co-transplantation of islets and MSCs. Increasing evidence indicates that co-transplantation of MSCs prolongs islet graft survival by locally secreted protective factors that reduce immune reactivity and promote vascularization, cell survival, and regeneration. MSC therapy may be a promising option for islet transplantation in patients with T1DM.

Cibinetide Protects Isolated Human Islets in a Stressful Environment and Improves Engraftment in the Perspective of Intra Portal Islet Transplantation

During intra-portal pancreatic islet transplantation (PITx), innate immune reactions such as the instant blood mediated inflammatory reaction (IBMIR) cause an immediate loss of islets. The non-hematopoietic erythropoietin analogue cibinetide has previously shown islet-protective effects in mouse PITx. Herein, we aimed to confirm cibinetide’s efficacy on human islets, and to characterize its effect on IBMIR. We cultured human islets with pro-inflammatory cytokines for 18 hours with or without cibinetide. ATP content and caspase 3/7 activity were measured. Dynamic glucose perfusion assay was used to evaluate islet function. To evaluate cibinetides effect on IBMIR, human islets were incubated in heparinized polyvinyl chloride tubing system with ABO compatible blood and rotated for 60 minutes to mimic the portal vein system. Moreover, human islets were transplanted into athymic mice livers via the portal vein with or without perioperative cibinetide treatment. The mice were sacrificed six days following transplantation and the livers were analyzed for human insulin and serum for human C-peptide levels. Histological examination of recipient livers to evaluate islet graft infiltration by CD11b+ cells was performed. Our results show that cibinetide maintained human islet ATP levels and reduced the caspase 3/7 activity during culture with pro-inflammatory cytokines and improved their insulin secreting capacity. In the PVC loop system, administration of cibinetide reduced the IBMIR-induced platelet consumption. In human islet to athymic mice PITx, cibinetide treatment showed an increased amount of human insulin in the livers and higher serum human C-peptide, while histological examination of the livers showed reduced infiltration of pro-inflammatory CD11b+ cells around islets grafts compared to the controls. In summary, Cibinetide protected isolated human islets in a pro-inflammatory milieu and reduced IBMIR related platelet consumption. It improved engraftment of human islets in athymic mice. The study confirms that cibinetide is a promising agent to be used in clinical PITx.

Preferable Transplant Site for Hepatocyte Transplantation in a Rat Model

Intraportal injection is regarded as the current standard procedure of hepatocyte transplantation (HTx). In islet transplantation, which shares many aspects with HTx, recent studies have clarified that instant blood-mediated inflammatory reaction (IBMIR), characterized by strong innate immune responses, can cause poor engraftment, so other transplant sites to avoid such a reaction have been established. Although IBMIR was reported to occur in HTx, few reports have evaluated alternative transplant sites for HTx. In this study, we sought to determine the optimum transplant site for HTx. Rat hepatocytes (1.0 × 107) were transplanted at the 9 transplant sites (intraportal (IPO), intrasplenic (IS), liver parenchyma, subcutaneous, intraperitoneal, renal subcapsular, muscle, inguinal subcutaneous white adipose tissue, and omentum) of analbuminemic rats. The serum albumin levels, immunohistochemical staining (albumin, TUNEL, and BrdU), and in vivo imaging of the grafts were evaluated. The serum albumin levels of the IPO group were significantly higher than those of the other groups (p < .0001). The BrdU-positive hepatocyte ratio of liver in the IS group (0.9% ± 0.2%) was comparable to that of the IPO group (0.9% ± 0.3%) and tended to be higher than that of the spleen in the IS group (0.5% ± 0.1%, p = .16). Considering the in vivo imaging evaluation and the influence of splenectomy, the graft function in the IS group may be almost entirely achieved by hepatocytes that have migrated to the liver. The present study clearly showed that the intraportal injection procedure is more efficient than other procedures for performing HTx

Characterizing and overcoming innate immunity in beta-cell replacement therapy

In clinical islet transplantation, the engrafted islet cell mass is a critical factor for maintaining graft function and transplant outcome. This review dissects components that contribute to an acute innate immune response, including ischemia-reperfusion injury, damage-associated molecular patterns, islet-derived cytokines (isletokines), and an instant blood-mediated inflammatory reaction. The cyclical amplification of immune cell response and islet inflammation can occur at each stage of the transplant process. As such, strategies to improve islet transplantation include minimizing islet and immune cell cytokine production prior to procurement; suppressing the pretransplant islet immune inflammatory response; inhibiting the instant blood-mediated inflammatory reaction in the liver; preventing an innate immune response by encapsulation and islet surface modification; targeting cytokine/chemokine secretion and innate immune cell infiltration; considering the optimal site for islet engraftment; blocking cytokine signaling in islets; and implementing anti-inflammatory strategies to improve islet transplantation. A large body of evidence indicates that targeting immune components at multiple steps throughout the transplant procedure can improve overall islet transplant outcomes.