Instant Blood-mediated Inflammatory Reaction Research Articles

Regulation of Inflammatory Response in Islet Transplantation

Islet cell transplantation is a developing treatment for patients suffering from severe Type-1 diabetes. The long-term insulin independence after islet cell transplantation has been difficult to achieve, and this has been linked to several factors. One of the major cause of poor long-term outcome is inflammation surrounding the islets. Inflammation in islets is caused art several stages, donor induced, during organ preservation, islet isolation stress, peri-transplant inflammation or instant blood mediated inflammatory reaction (IBMIR), and post-transplant hypoxia. In addition to inflammation, auto/allo-immune attack causes rejection and the toxicity of the immunosuppressive agents used can also affect islet transplant outcomes. In this review, we will summarize the various inflammatory processes that occur during islet transplantation along with past and previous approaches used to reduce inflammation in pre-clinical and clinical studies.

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating.

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

Layer-by-Layer Heparinization of the Cell Surface by Using Heparin-Binding Peptide Functionalized Human Serum Albumin.

Layer-by-layer heparinization of therapeutic cells prior to transplantation is an effective way to inhibit the instant blood-mediated inflammatory reactions (IBMIRs), which are the major cause of early cell graft loss during post-transplantation. Here, a conjugate of heparin-binding peptide (HBP) and human serum albumin (HSA), HBP-HSA, was synthesized by using heterobifunctional crosslinker. After the first heparin layer was coated on human umbilical vein endothelial cells (HUVECs) by means of the HBP-polyethylene glycol-phospholipid conjugate, HBP-HSA and heparin were then applied to the cell surface sequentially to form multiple layers. The immobilization and retention of heparin were analyzed by confocal microscopy and flow cytometry, respectively, and the cytotoxity of HBP-HSA was further evaluated by cell viability assay. Results indicated that heparin was successfully introduced to the cell surface in a layer-by-layer way and retained for at least 24 h, while the cytotoxity of HBP-HSA was negligible at the working concentration. Accordingly, this conjugate provides a promising method for co-immobilization of heparin and HSA to the cell surface under physiological conditions with improved biocompatibility.

Display of CD47 protein on pancreatic islet grafts improves engraftment following intraportal transplantation

Abstract Intraportal islet transplantation is a curative therapy for type 1 diabetes. However, early islet engraftment is hampered by a thrombotic and inflammatory reaction defined as an instant blood-mediated inflammatory reaction (IBMIR). The CD47 molecule is an important regulator of IBMIR, by blocking phagocytosis. We hypothesized that the transient display of the CD47 protein on the surface of islet grafts may prevent early islet destruction by IBMIR following intraportal transplantation. We generated a chimeric CD47 fused with a modified streptavidin (SA-CD47). SA-CD47 was transiently displayed on the surface of rat splenocytes that had been modified with biotin taking advantage of the strong affinity between biotin and streptavidin (SA). SA-CD47 protein was also effectively displayed on the surface of biotinylated islets. Importantly, in a syngeneic marginal mass intraportal islet transplantation model, 7/8 streptozotocin-diabetic C57BL/6 mice transplanted with 125 SA-CD47 engineered islets established euglycemia long term (>80 days). In marked contrast, only 1/7 mice achieved euglycemia when transplanted with SA-engineered islet grafts. The engrafted islets in the SA-CD47 group had a comparable response to naïve mice in the intraperitoneal glucose tolerance test. Flow cytometric and qRT-PCR analyses of liver tissues did not reveal significant changes in cellular and molecular immune responses between SA-CD47 and SA-engineered control groups, suggesting that inhibition of islet phagocytosis as underlying mechanism of islet engraftment and survival. Collectively, these results demonstrate the utility of SA-CD47 as a regulator of IBMIR for prevention of early islet graft loss following intraportal transplantation

Polymeric nano-shielded islets with heparin-polyethylene glycol in a non-human primate model

Intraportal pancreatic islet transplantation incurs huge cell losses during its early stages due to instant blood-mediated inflammatory reactions (IBMIRs), which may also drive regulation of the adaptive immune system. Therefore, a method that evades IBMIR will improve clinical islet transplantation. We used a layer-by-layer approach to shield non-human primate (NHP) islets with polyethylene glycol (nano-shielded islets, NSIs) and polyethylene glycol plus heparin (heparin nano-shielded islets; HNSIs). Islets ranging from 10,000 to 20,000 IEQ/kg body weight were transplanted into 19 cynomolgus monkeys (n = 4, control; n = 5, NSI; and n = 10, HNSI). The mean C-peptide positive graft survival times were 68.5, 64 and 108 days for the control, NSI and HNSI groups, respectively (P = 0.012). HNSI also reduced the factors responsible for IBMIR in vitro. Based on these data, HNSIs in conjunction with clinically established immunosuppressive drug regimens will result in superior outcomes compared to those achieved with the current protocol for clinical islet transplantation.

The Optimization of the Hepatocyte Surface Modification Procedures in Terms of Heparin and Apyrase for Improving Hepatocyte Engraftment

Background The rapid destruction of hepatocyte grafts immediately after transplantation has hampered the application of this procedure as a clinical treatment. The instant blood-mediated inflammatory reaction (IBMIR), which is characterized by the activation of both coagulation and complement cascades, is a plausible underlying cause for this poor engraftment. In pancreatic islet transplantation, which shares many aspects with hepatocyte transplantation, it has been reported that islet surface heparinization efficiently inhibits the IBMIR without increasing the bleeding risk. In the present study, we tried to optimize the hepatocyte surface modification procedures in terms of heparin and apyrase for improving hepatocyte engraftment. Methods Rat hepatocytes were isolated using a modified two-step collagenase perfusion technique and then purified by Percoll density gradient centrifugation. Hepatocytes (2.0 × 106) were mixed with heparin-binding peptide (CNSAHRTRGRQRS)-poly (ethylene glycol)-conjugated phospholipid (HBP-PEG-lipid) solution or Maleimide-PEG-lipid solution, and then a solution of heparin conjugate (Corline AB, Uppsala, Sweden) or apyrase-SH was added for 10 minutes. All of these procedures were performed at either room temperature (RT) or 4 °C using either phosphate-buffered saline (PBS) or optimized culture medium for hepatocytes with or without fetal bovine serum (FBS). The hepatocyte viability was evaluated by a trypan blue exclusion assay. The efficiency of the hepatocyte surface modification procedures was evaluated by detecting AlexaFluor 488 under fluorescence microscopy. Results Regarding both heparin and apyrase, the surfaces of hepatocytes were sufficiently coated in all groups. The hepatocyte viability prior to the surface modification procedures was 94.09±2.18%. When PBS was used for the procedures, the viability of heparin-coated hepatocytes was 26.78±5.27% at RT and 63.53±5.95% at 4 °C. Likewise, the viability of apyrase-coated hepatocytes was 33.04±11.37% at RT and 60.43±6.63% at 4 °C. Of particular note, the viability of non-coated hepatocytes was 57.03±17.25% at RT and 66.65±1.28% at 4 °C, suggesting that the hepatocyte viability was substantially damaged during the preservation period rather than due to the coating chemicals per se. When using optimized culture medium for hepatocytes at 4 °C, the viability of heparin-coated and apyrase-coated hepatocytes markedly increased to 85.76±0.74% and 85.96±3.51%, respectively, under the supplementation of FBS, whereas the viability of heparin-coated hepatocytes remained at 73.79±7.42% without FBS. Conclusions The present study revealed that hepatocyte surface modification in terms of heparin and apyrase could be efficiently performed with minimal damage to the hepatocytes by introducing an optimized culture medium for hepatocytes including FBS, instead of carrying out the standard procedure (PBS), at 4 °C.

Selection of Tissue Factor-Deficient Cell Transplants as a Novel Strategy for Improving Hemocompatibility of Human Bone Marrow Stromal Cells.

Intravascular transplantation of tissue factor (TF)-bearing cells elicits an instant blood-mediated inflammatory reaction (IBMIR) resulting in thrombotic complications and reduced engraftment. Here we studied the hemocompatibility of commonly used human white adipose tissue (WAT), umbilical cord (UC) and bone marrow stromal cells (BMSC) and devised a possible strategy for safe and efficient stromal cell transplantation.Methods: Stromal cell identity, purity, and TF expression was tested by RTQ-PCR, flow cytometry and immunohistochemistry. Pro-coagulant activity and fibrin clot formation/stabilization was measured In Vitro by viscoelastic rotational plasma-thromboelastometry and in vivo by injecting sorted human stromal cells intravenously into rats. The impact of TF was verified in factor VII-deficient plasma and by sort-depleting TF/CD142+ BMSC.Results: We found significantly less TF expression by a subpopulation of BMSC corresponding to reduced pro-coagulant activity. UC and WAT stroma showed broad TF expression and durable clotting. Higher cell numbers significantly increased clot formation partially dependent on coagulation factor VII. Depleting the TF/CD142+ subpopulation significantly ameliorated BMSC's hemocompatibility without affecting immunomodulation. TF-deficient BMSC did not produce thromboembolism in vivo, comparing favorably to massive intravascular thrombosis induction by TF-expressing stromal cells.Conclusion: We demonstrate that plasma-based thromboelastometry provides a reliable tool to detect pro-coagulant activity of therapeutic cells. Selecting TF-deficient BMSC is a novel strategy for improving cell therapy applicability by reducing cell dose-dependent IBMIR risk. The particularly strong pro-coagulant activity of UC and WAT preparations sounds an additional note of caution regarding uncritical systemic application of stromal cells, particularly from non-hematopoietic extravascular sources.

Early barriers to neonatal porcine islet engraftment in a dual transplant model.

Porcine islet xenografts have the potential to provide an inexhaustible source of islets for β cell replacement. Proof-of-concept has been established in nonhuman primates. However, significant barriers to xenoislet transplantation remain, including the poorly understood instant blood-mediated inflammatory reaction and a thorough understanding of early xeno-specific immune responses. A paucity of data exist comparing xeno-specific immune responses with alloislet (AI) responses in primates. We recently developed a dual islet transplant model, which enables direct histologic comparison of early engraftment immunobiology. In this study, we investigate early immune responses to neonatal porcine islet (NPI) xenografts compared with rhesus islet allografts at 1hour, 24hours, and 7days. Within the first 24hours after intraportal infusion, we identified greater apoptosis (caspase 3 activity and TUNEL [terminal deoxynucleotidyl transferase dUTP nick end labeling])-positive cells) of NPIs compared with AIs. Macrophage infiltration was significantly greater at 24hours compared with 1hour in both NPI (wild-type) and AIs. At 7days, IgM and macrophages were highly specific for NPIs (α1,3-galactosyltransferase knockout) compared with AIs. These findings demonstrate an augmented macrophage and antibody response toward xenografts compared with allografts. These data may inform future immune or genetic manipulations required to improve xenoislet engraftment.

Enhanced effect of human mesenchymal stem cells expressing human TNF-αR-Fc and HO-1 gene on porcine islet xenotransplantation in humanized mice.

Porcine islet xenotransplantation is considered an attractive alternative treatment for type 1 diabetes mellitus. However, it is largely limited because of initial rejection due to Instant Blood-Mediated Inflammatory Reaction (IBMIR), oxidative stress, and inflammatory responses. Recently, soluble tumor necrosis factor-ɑ receptor type I (sTNF-αR) and heme oxygenase (HO)-1 genes (HO-1/sTNF-αR) have been shown to improve the viability and functionality of porcine islets after transplantation. In this study, genetically modified mesenchymal stem cells (MSCs) expressing the HO-1/sTNF-αR genes (HO-1/sTNF-αR-MSC) were developed using an adenoviral system, and porcine islet viability and function were confirmed by in vitro tests such as GSIS, AO/PI, and the ADP/ATP ratio after coculturing with HO-1/sTNF-αR-MSCs. Subsequently, isolated porcine islets were transplanted underneath the kidney capsule of diabetic humanized mice without MSCs, with MSCs or with HO-1/sTNF-αR-MSCs. According to the results, the HO-1/sTNF-αR-MSC-treated group exhibited improved survival of porcine islets and could reverse hyperglycemia more than porcine islets not treated with MSCs or islets cotransplanted with MSCs. Moreover, the HO-1/sTNF-αR-MSC group maintained its morphological characteristics and the insulin secretion pattern of transplanted porcine islets similar to endogenous islets in immunocompetent humanized mice. Our results suggest that HO-1/sTNF-αR-MSCs are efficient tools for porcine islet xenotransplantation, and this study may provide basic information forpre-clinical animal models and future clinical trials of porcine islet xenotransplantation.

Culturing with modified EGM2 medium enhances porcine neonatal islet-like cell clusters resistance to apoptosis in islet xenotransplantation.

Neonatal pig islet-like cell clusters (NICC) are an attractive source of insulin-producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood-mediated inflammatory reaction remains to be overcome. EGM2 medium depleted with hydrocortisone and supplemented with 50mmol/L isobutylmethylxanthine, 10mmol/L nicotinamide, and 10mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM2 or control Ham's F-10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO/EB staining and FACS. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon gene expression were measured by real-time PCR. Tubing loops model and TUNEL assay were performed to confirm the apoptosis-resistant ability of NICC cultured with modified EGM2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting. Compared with Ham's F-10 medium, culturing NICC with EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR. Moreover, EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F-10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti-apoptotic Bcl-2 family member Mcl-1. Culturing NICC with EGM2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation.

Mechanism for the Instant Blood-Mediated Inflammatory Reaction in Rat Islet Transplantation

ObjectiveThe purpose of this study was to investigate the mechanisms involved in the development of instant blood-mediated inflammatory reaction (IBMIR) triggered by pancreatic islet transplantation in an in vitro system. Materials and MethodsPancreatic islets were prepared and blood was taken from male and female Wistar rats. An in vitro heparinized tubing loop model on a rocking device that was placed in a 37°C incubator was used to generate blood flow inside the loops. Rat blood was added to the device, and after a 60-minute incubation period, 800 islet equivalents were added to the system. Rat blood samples were taken for immediate hematologic analysis before circulation, before the addition of islets, and 5, 15, 30, and 60 minutes after the addition of islets. After 60 minutes, the blood in the tubing was filtered, and the filtrate was stained using hematoxylin and eosin for microscopic analysis. ResultsFollowing the addition of islets, the counts of platelets, white blood cells, and mononuclear cells were significantly decreased at 60 min (P < .05), and the plasma levels of thrombin-antithrombin complexes and complement C3a were significantly increased (P < .05). At 60 minutes after introduction of the islets, the thrombi and tissue in the remaining blood in the device consisted of a few islets with serious structural damage and incomplete capsules, and there were many microthrombi with a large number of infiltrating neutrophils around the islets. ConclusionOur in vitro model of IBMIR was characterized by platelet consumption, activation of the coagulation and complement cascade systems, and leukocytic infiltration.

MRI-sensitive contrast agent with anticoagulant activity for surface camouflage of transplanted pancreatic islets.

Pancreatic islet implantation in the liver is a promising approach for diabetes therapy. However, 70% of the islet mass fails to be engrafted in the liver due to the instant blood-mediated inflammatory reactions (IBMIR) resulting from direct contact between islet cells and the bloodstream. To overcome this issue, direct monitoring is very important for establishing prognosis after islet cell therapy. Here we established a new type of MR contrast agent with anticoagulant activity via heparin-immobilized superparamagnetic iron oxide (HSPIO). The HSPIO was chemically conjugated onto islet surface exvivo without damage of their viability and functionality. The conjugated HSPIO nanoparticles onto islet surface could attenuate IBMIR invitro and invivo. The HSPIO-conjugated islets could cure the blood glucose levels of diabetes animals after implantation. In addition, the HSPIO nanoparticles were well maintained on the transplanted islets for a long time during modulation of inflammation. Also, they allowed for stable visualization of the implanted islet cells for more than 150 days without reduction of the MRI signal. Furthermore, when HSPIO itself was intraportally injected, it was rapidly eliminated without accumulation in the liver, suggesting that HSPIO nanoparticles could only track the immobilized islet. Collectively, this HSPIO nanoparticle having MRI sensitivity and anticoagulant activity could be utilized for successful islet implantation.

Activation of Liver-Resident DX5 Negative NK Cells after Intraportal Islet Transplantation Prevent the Engraftment of Secondary Transplanted Islets

It has been recently reported that liver-resident DX5− natural killer (NK) cells have potent memory functions, which include differentiation into a long-lived memory subset and robust antigen-specific expansion to a secondary challenge. We have previously demonstrated that liver NK cells play a pivotal role in islet graft loss during the early phase after intraportal syngeneic islet transplantation (IT). In this study, we investigated the mechanism of liver-resident DX5− NK cells activation after IT and the influence of primary IT on the engraftment of secondarily transplanted islets. To evaluate the mechanism of NK cells activation early after IT, liver NK cells from C57BL/6 mice were cultured for 24 h in the presence of TNFα, IFNγ, and IL-1β, mimicking instant blood-mediated inflammatory reaction. Furthermore, we established a multiple IT model for evaluating the memory functions of NK cells in vivo; 400 islets were secondarily transplanted following primary IT with 200 islets. Surface molecules on liver NK cells were analyzed to evaluate the phenotypical and functional alteration of liver NK cells from the early to the late phase after IT. The population of DX5− NK cells significantly expanded 2.4 times compared to naive condition (p < 0.01) after the cytokine stimulation dominantly associated with TNFα, and the significant increase of both CD69 and CXCR3 expression on liver NK cells was observed (24 h/fresh ratio; 2.0, p < 0.01; 1.5, p < 0.01, respectively). In in vivo assay, the population of DX5− NK cells also promptly expanded and the expression of CD69, TRAIL, and CXCR3 on liver NK cells were significantly increased at 24 h after IT, meanwhile this phenomenon was restored by the administration of anti-TNFα antibody. At the late phase after IT, a significant and persistent increase of DX5− NK cells, expression of CD69, TRAIL, and CXCR3 on liver NK cells was observed compared to naive condition (35 d/0 d ratio; 2.2, p < 0.01, 2.3, p < 0.01, 3.7, p < 0.01, 2.0, p < 0.05, respectively). The syngeneic 400 islets of primary IT mostly achieved normoglycemia in diabetic mice, whereas the same mass of secondary IT failed to induce normoglycemia in the recipients received syngeneic 200 islets of primary IT. Our findings indicate that the liver-resident DX5− NK cells significantly and persistently expanded even after syngeneic IT, and those memory-like NK cells may target both of originally engrafted islets and even secondary transplanted islets.

Pancreatic islet surface bioengineering with a heparin-incorporated starPEG nanofilm

Cell surface engineering could protect implanted cells from host immune rejections while modify the cellular landscape for better post-transplantation graft function and survival. Islet transplantation is considered the most promising therapeutic option with the potential to cure diabetes. Current approach to improve clinical efficacy of pancreatic islet transplantation is alginate encapsulation. However, disappointing outcomes have been reported in clinical trials due to larger islet size resulted by encapsulation and alginate-elicited host immune responses. We have developed an ultrathin nanofilm of starPEG with incorporated heparin (Hep-PEG) that binds covalently to the amine groups of islet surface membrane via its N-hydroxysuccinimide groups. The Hep-PEG nanocoating elicited minimal alteration on islet volume in culture. Hep-PEG-coated islets exhibited robust islet viability accompanied by uncompromised islet insulin secretory function. Instant blood-mediated inflammatory reaction was also reduced by Hep-PEG islet coating, accompanied by enhanced intra-islet revascularization. In addition, despite its semi-permeability, Hep-PEG islet coating promoted the survival of islets exposed to pro-inflammatory cytokines. Considering that inflammation and hypoxia are primary causes of immediate cell loss for cell therapy, the Hep-PEG nanofilm represents a viable approach for cell surface engineering which would improve the clinical outcome of cell therapies.

Intraportal islet transplantation: the impact of the liver microenvironment.

The portal vein remains the preferred site for pancreatic islet transplantation due to its easy access and low morbidity. However, despite great progress in isolation and transplantation protocols over the past few years, it is still associated with the early loss of some 50-70% of transplanted islets. The complex liver microenvironment itself presumably plays an important role in this loss. The present review focuses on the specifics of the liver microenvironment, notably the localized hepatic ischemia/reperfusion injury following transplantation, the low oxygenation of the portal vein, the instant blood-mediated inflammatory reaction, the endogenous liver immune system, and the gut-liver axis, and how they can each have an impact on the transplanted islets. It identifies the potential, or already applied, clinical interventions for improving intraportal islet survival, and pinpoints those promising areas still lacking preclinical research. Future interventions on clinical intraportal islet transplantation need to take into account the global context of the liver microenvironment, with multi-point interventions being most likely to improve early islet survival and engraftment.

Human Recombinant Antithrombin (ATryn®) Administration Improves Survival and Prevents Intravascular Coagulation after Intraportal Islet Transplantation in a Piglet Model

Human islet transplantation is a viable treatment option for type 1 diabetes mellitus (T1DM). However, pancreatic islet inflammation after transplantation induced by innate immune responses is likely to hinder graft function. This is mediated by incompatibility between islets and the blood interface, known as instant blood-mediated inflammatory reaction (IBMIR). Herein we hypothesized that portal venous administration of islet cells with human recombinant antithrombin (ATryn ® ), a serine protease inhibitor (serpin), which plays a central role in the physiological regulation of coagulation and exerts indirect anti-inflammatory activities, may offset coagulation abnormalities such as disseminated intravascular coagulation (DIC) and IBMIR. The current prospective, randomized experiment was conducted using an established preclinical pig model. Three groups were constituted for digested pancreatic tissue transplantation (0.15 ml/kg): control, NaCl 0.9% (n = 7); gold standard, heparin (25 UI/kg) (n = 7); and human recombinant ATryn® (500 UI/kg) (n = 7). Blood samples were collected over time (T0 to 24 h), and biochemical, coagulation, and inflammatory parameters were evaluated. In both the control and heparin groups, one animal died after a portal thrombosis, while no deaths occurred in the ATryn®-treated group. As expected, islet transplantation was associated with an increase in plasma IL-6 or TNF-α levels in all three groups. However, DIC was only observed in the control group, an effect that was suppressed after ATryn® administration. ATryn® administration increased antithrombin activity by 800%, which remained at 200% for the remaining period of the study, without any hemorrhagic complications. These studies suggest that coadministration of ATryn® and pancreatic islets via intraportal transplantation may be a valuable therapeutic approach for DIC without risk for islets and subjects.

α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction.

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon.

Control of IBMIR Induced by Fresh and Cryopreserved Hepatocytes by Low Molecular Weight Dextran Sulfate versus Heparin

Rapid destruction of hepatocytes after hepatocyte transplantation has hampered the application of this procedure clinically. The instant blood-mediated inflammatory reaction (IBMIR) is a plausible underlying cause for this cell loss. The present study was designed to evaluate the capacity of low molecular weight dextran sulfate (LMW-DS) to control these initial reactions from the innate immune system. Fresh and cryopreserved hepatocytes were tested in an in vitro whole-blood model using ABO-compatible blood. The ability to elicit IBMIR and the capacity of LMW-DS (100 μg/ml) to attenuate the degree of activation of the cascade systems were monitored. The effect was also compared to conventional anticoagulant therapy using unfractionated heparin (1 IU/ml). Both fresh and freeze–thawed hepatocytes elicited IBMIR to the same extent. LMW-DS reduced the platelet loss and maintained the cell counts at the same degree as unfractionated heparin, but controlled the coagulation and complement systems significantly more efficiently than heparin. LMW-DS also attenuated the IBMIR elicited by freeze–thawed cells. Therefore, LMW-DS inhibits the cascade systems and maintains the cell counts in blood triggered by both fresh and cryopreserved hepatocytes in direct contact with ABO-matched blood. LMW-DS at a previously used and clinically applicable concentration (100 μg/ml) inhibits IBMIR in vitro and is therefore a potential IBMIR inhibitor in hepatocyte transplantation.

Mesenchymal stem cells to promote islet transplant survival.

Mesenchymal stromal cells (MSCs) are adult stromal cells with therapeutic potential in allogeneic islet transplantation for type 1 diabetes patients. The process of islet isolation alone has been shown to negatively impact islet survival and function in vivo. In addition, insults mediated by the instant blood-mediated inflammatory reaction, hypoxia, ischemia and immune response significantly impact the islet allograft post transplantation. MSCs are known to exert cytoprotective and immune modulatory properties and thus are an attractive therapeutic in this context. Herein, the recent progress in the field of MSC therapy in islet transplantation is discussed. MSC can promote islet survival and function in vivo. Importantly, studies have shown that human MSC donors have differential abilities in promoting islet regeneration/survival. Recently, several biomarkers associated with MSC islet regenerative capacity have been identified. Expressions of Annexin A1, Elastin microfibril interface 1 and integrin-linked protein kinase are upregulated in MSC displaying protective effects on islet survival and function in vivo. The discovery of biomarkers associated with MSC therapeutic efficacy represents an important step forward for the utilization of MSC therapy in islet transplantation; however, much remains to be elucidated about the mechanisms utilized by MSC in protection against transplanted islet loss, autoimmune-mediated and alloimmune-mediated rejection.