P. Phillips

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.

Adoptive Transfer With In Vitro Expanded Human Regulatory T Cells Protects Against Porcine Islet Xenograft Rejection via Interleukin-10 in Humanized Mice

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ−/− mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

The Importance of Tissue Factor Expression by Porcine NICC in Triggering IBMIR in the Xenograft Setting

Background. In islet transplantation, tissue factor (TF) has been reported to be involved in triggering the instant blood-mediated inflammatory reaction (IBMIR), which causes early massive loss of islets transplanted intraportally. TF is synthesized and secreted by several cell sources including islets and inflammatory cells such as neutrophils, monocytes, and platelets. In this study, we investigated whether xenografts-mediated IBMIR could be inhibited by selectively inhibiting TF production by islets using small interfering RNA (siRNA)-mediated TF gene knockdown. Methods. Porcine neonatal islet cell clusters (NICC) were transfected with siRNA specific for TF or a nonspecific siRNA. TF gene and protein expression were analyzed by real-time polymerase chain reaction and fluorescence-activated cell sorting, respectively. The effect of TF knockdown on IBMIR was evaluated using an in vitro tubing loop model of human blood-NICC interactions. Results. TF siRNA transfection of NICC resulted in reduced TF gene and protein expression. TF siRNA transfected NICC showed a significant reduction in the formation of blood clots, platelet activation, thrombin generation, and complement activation after exposure to human ABO compatible blood in vitro. In addition, there was reduced neutrophil infiltration within blood clots containing TF siRNA transfected NICC. Conclusions. TF expression on porcine NICC is an important initiator of IBMIR in islet xenotransplantation. This study identifies porcine TF as a potential target for inhibiting this response.

Long-term cultured neonatal islet cell clusters demonstrate better outcomes for reversal of diabetes: in vivo and molecular profiles

Porcine neonatal islet‐like cell clusters (NICC) are being considered as a source of β‐cell replacement. However, the lag time to full function due to hormonal immaturity remains a problem. This study aimed to determine whether time in culture was important for NICC function in vivo.

Anti-CD2 producing pig xenografts effect localized depletion of human T cells in a huSCID model

We investigated whether graft produced anti‐human CD2, mediated by adenovirus (Adv) transduction of pig neonatal islet cell clusters (pNICC), would protect xenografts in a humanized mouse model from immune attack and whether such immunosuppression would remain local.

Pancreatic Transdifferentiation in Porcine Liver Following Lentiviral Delivery of Human Furin–Cleavable Insulin

Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic β-cells. Gene therapy is one strategy being actively explored to cure TID by affording non-β-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)-diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some β-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 10(9) transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several β-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed. Triple immunofluorescence showed extensive insulin expression, as was previously observed in our studies with rodents. Additionally, a small amount of glucagon and somatostatin protein expression was seen. Collectively, these data indicate that pancreatic transdifferentiation of the liver tissue had occurred. Our data suggest that this regimen may ultimately be used clinically to cure TID, however more work is required to replicate the successful reversal of diabetes in increased numbers of pigs.

A Humanized Mouse Model to Study Human Immune Response in Xenotransplantation

BACKGROUND A major barrier to the clinical application of xenotransplantation as a treatment option for patients is T cell-mediated rejection. Studies based on experimental rodent models of xenograft tolerance or rejection in vivo have provided useful information about the role of T cell immune response in xenotransplantation. However not all observations seen in rodents faithfully recapitulate the human situation. This study aimed to establish a humanized mouse model of xenotransplantation, which mimics xenograft rejection in the context of the human immune system. METHODS NOD-SCID IL2rgamma-/- mice were transplanted with neonatal porcine islet cell clusters (NICC) followed by reconstitution of human peripheral blood mononuclear cells (PBMC). Human leukocyte engraftment and islet xenograft rejection were confirmed by flow cytometric and histological analyses. RESULTS In the absence of human PBMC, porcine NICC transplanted into NOD-SCID IL2rgamma-/- mice revealed excellent graft integrity and endocrine function. Human PBMC demonstrated a high level of engraftment in NOD-SCID IL2rgamma-/- mice. Reconstitution of NICC recipient NOD-SCID IL2rgamma-/- mice with human PBMC led to the rapid destruction of NICC xenografts in a PBMC number-dependent manner. CONCLUSIONS Human PBMC-reconstituted NOD-SCID IL2rgamma-/- mice provide an ideal model to study human immune responses in xenotransplantation. Studies based on this humanized mouse model will provide insight for improving the outcomes of clinical xenotransplantation.

Restoration of Function with Human Islets in Humanized Diabetic Mice with Subsequent Rejection of Islet Allografts by Allogeneic Engrafted Human T Cells: 967

Hu M.1, Hawthorne W.J.1, Burns H.1, Davies S.1, Zhang G.Y.2, Hawkes J.1, Wu J.1, Jimenez-Vera E.1, Williams L.1, Patel T.1, Phillips P.1, Hor K.1, Wang Y.M.2, Watson D.2, Jin X.1, Qian Y.W.1, Yi S.1, Alexander S.I.2, O‘Connell P.J.1 1University of Sydney, Centre for Transplant and Renal Research, Westmead Millennium Institute, Westmead, Australia, 2University of Sydney, Centre for Kidney Research, Children‘s Hospital at Westmead, Westmead, Australia

SIRNA-MEDIATED TISSUE FACTOR KNOCKDOWN IN NEONATAL PORCINE ISLET CELL CLUSTERS RESULTED IN SUPPRESSION OF IBMIR IN VITRO: 1516

Introduction: Instant blood-mediated inflammatory reaction (IBMIR), characterized by a rapid binding, and activation of platelets to the islet surface, infiltration of the islets by leukocytes, and activation of the coagulation and complement systems, causes early loss of islets transplanted intraportally and has been recognized as one of hurdles to be overcome for successful islet transplantation. Tissue factor (TF) is an important initiator of the blood clotting system. In this study we investigated whether suppressed expression of tissue factor in neonatal porcine islet cell clusters (NICC) can inhibit IBMIR in an in vitro tubing loop model of human blood-NICC interaction. Methods: NICC were transfected with or without TF siRNA or nonspecific siRNA by lipofectamine 2000. Transfected NICC were then analyzed for TF gene and protein expression by real-time PCR and FACS respectively. The effect of TF knockdown in NICC on IBMIR was evaluated in the tubing loop system. Clots were collected, measured and sectioned for immunochemical examination. The degree of neutrophil infiltration was determined by scoring neutrophils surrounding individual NICC in clots. Platelets, leukocytes and neutrophils were counted by using Beckman Coulter ACT. Results: TF siRNA transfection of NICC resulted in substantially reduced expression of both TF gene and protein expression. Nonspecific siRNA had no effect. When TF siRNA transfected NICC were incubated with human blood there was reduced clot weight (0.380.04g vs 0.700.02g, p< 0.01) and reduced consumption of platelets (71±12ï‚ ́109/L vs 9±1ï‚ ́109/L, p< 0.01), leukocytes (3.42±0.59ï‚ ́109/L vs 2.60±0.23ï‚ ́109/L, p< 0.05) and neutrophils (1.73±0.09ï‚ ́109/L vs 1.08±0.04ï‚ ́109/L, p<0.05) when compared to that detected in the tubing loop with non-transfected NICC. In addition, immunohistochemical examination showed significant reduction in neutrophil infiltration in clots collected from the tubing loop system with TF siRNA transfected NICC and human blood compared with that found in the clots from the tubing loop with non-transfected or nonspecific siRNA transfected NICC and human blood. Conclusion: This study supports the hypothesis that NICC TF expression is an important initiator of IBMIR.

A PATHWAY FOR HUMAN ISLET PROTEASE-ACTIVATED RECEPTORS (PARS) IN TISSUE FACTOR MEDIATED IMMEDIATE BLOOD INFLAMMATORY REACTION.: 230

Introduction: Clinical islet transplantation requires the transplantation of multiple islet preparations to achieve successful transplant outcomes. This is due to islet cell loss through apoptotic and other mechanisms which may claim as much as 60% of the islet mass. One of the major contributing factors is when the islet comes into contact with blood and Immediate Blood Mediated Inflammatory Reaction (IBMIR) occurs. The biology of human islets at the signalling and molecular level is poorly understood, less is known how islet isolation transplantation and inflammation impacts on islet survival. Protease-activated receptors (PARs) comprise a family of G-proteincoupled receptors with a unique proteolytic activation mechanism. PARs regulate a broad range of cellular functions and are involved in the pathogenesis of inflammatory disorders. Moreover, PAR1 and PAR2 activation in the endothelium shifts it toward a prothrombotic condition. Thrombin seems to be the main physiological activator of these receptors but several other proteases can contribute to their function. The aim of this study was to assess the factors involved in the signalling events underlying tissue factor (TF) expression elicited by PAR1 and PAR2 in islet cells, and to investigate their mechanisms. Methods: Human Islets prepared for clinical transplantation were exposed to specific PAR1 and PAR2 agonist peptides (PAR-APs). TF expression and protein expression was determined by RT-PCR and western blot assays analysis and measurement of procoagulant activity was performed by clotting assays. Results: PAR 1 and PAR2 mRNA levels were detected in resting islets, PAR2 expression was 20% that of PAR1. Both PAR1 and PAR2-APs induced TF activity in a timeand concentration-dependent manner. PAR1-AP was more effective that PAR2-AP. Both PAR-APs increased TF mRNA levels. TF up-regulation by PAR-APs was mediated by ERK1/2 and p38 MAPK activation, with PAR-APs promoting a rapid and transient increase in ERK1/2 and p38 activity, measured by an increase in phosphoractivity. Furthermore, MEK and p38 inhibitors significantly reduced TF activity induced by PAR-APs. Evidence for a novel PAR1 receptor signalling that flows through G protein and requires cooperation between protein kinase A (PKA) and NF-κB has been reported. The relevance of this signalling module was therefore, addressed in islets exposed to PAR-APs. A Gαi/o subunit and a PKA inhibitor selectively prevented TF activity in cells incubated with PAR1-AP. The pivotal role of the signalling module involving the association between PKA and NF-κB was further investigated by targeting NF-κB activation by means of a specific NF-κB inhibitor peptide or by a proteasome inhibitor. Both these approaches prevented TF induction by PAR1-AP. Conclusions: Our data identify ERK1/2 and MAPK as key signalling pathways triggered by PAR1 and PAR2 engagement in human islets and show that downstream from receptor activation occur cascades that are mechanistically coupled to increased procoagulant activity. We show that a signalling module flowing through Gαi/o subunit, PKA and NF-κB is involved in TF induction by PAR-AP. The elucidation of pathways downstream from PAR1 and PAR2 activation may be helpful in the design of pharmacological strategies to control the procoagulant and inflammatory effect of IBMIR.