<p>During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. </p><p>The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. </p><p>In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization.</p><p>Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process.</p><p>The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.</p>

During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization. Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process. The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.

<p>Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. </p><p>An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. </p><p>In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified.</p><p>The thrombin inhibitor melagatran completely blocked the IBMIR in an <i>in vitro</i> tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. </p><p>These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.</p>

<p>Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity.</p><p>In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation<i> in vivo</i>. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF <i>in vitro</i> efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated <i>in vivo</i> in patients transplanted with isolated islets.</p><p>There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets <i>in vitro</i> and<i> in vivo</i>, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials. </p>

Clinical islet transplantation is an emerging procedure to cure type 1 diabetes. The graft is implanted by infusion into the liver through the portal vein. A major obstacle that still needs to be overcome is the requirement for islets from multiple donors to achieve insulin independence. An innate inflammatory reaction, the IBMIR, is elicited when islets are exposed to blood. The IBMIR has been described as a clotting reaction culminating in disruption of islet morphology and is a plausible cause for loss of tissue during the early post-transplant period. In this thesis, the underlying mechanisms of the IBMIR were characterized. The IBMIR was for the first time demonstrated in patients undergoing an islet transplant, and a number of clinically applicable strategies to limit this reaction were identified. The thrombin inhibitor melagatran completely blocked the IBMIR in an in vitro tubing blood loop system, indicating that thrombin is the driving force in the reaction. Interestingly, islets were shown to produce and secrete tissue factor (TF), the physiological trigger of coagulation. Inactivated FVIIa, a specific inhibitor of TF, successfully blocked initiation of the IBMIR. An alternative approach to limit the IBMIR was to pre-treat islets in culture prior to transplantation. Nicotinamide added to the culture medium effectively decreased the level of TF in human islets. Infiltration of immune cells, also a part of the IBMIR, was characterized in detail. The predominant cell types infiltrating the islets were neutrophilic granulocytes and, to a lesser degree, monocytes. Both cell types may exert direct cytotoxic effects, and the antigen-presenting monocytes may also be important for directing the specific immune system to the site of inflammation. These findings have provided new insight into the nature of the IBMIR and offer several new strategies to improve the outcome of clinical islet transplantation.

Intraportal transplantation of isolated islets of Langerhans is a procedure approaching clinical acceptance as a treatment for patients with type I diabetes mellitus. One major problem with this treatment is that large amounts of cells are lost at the time of infusion into the portal vein, resulting in a low level of engraftment of the islets. One likely explanation for this loss is the instant blood-mediated inflammatory reaction (IBMIR), a thrombotic/inflammatory reaction occurring when islets come in contact with blood. The IBMIR is characterized by coagulation and complement activation, leading to platelet consumption, leukocyte infiltration of the islets, and disruption of islet integrity. In this thesis, the IBMIR is shown to be triggered by tissue factor (TF), the main initiator of blood coagulation in vivo. TF is expressed in two forms by the endocrine cells of the pancreas, a full-length membrane-bound and an alternatively spliced soluble form. Blocking TF in vitro efficiently reduces the macroscopic clotting, expression of coagulation activation markers, and leukocyte infiltration. This blockade can be achieved by adding either an active site-specific anti-TF antibody or site-inactivated FVIIa that competes with active FVIIa in the blood. TF may be secreted from the islets, since it is colocalized with insulin and glucagon in their granules. The IBMIR has also been demonstrated in vivo in patients transplanted with isolated islets. There are two ways to block the IBMIR in transplantation: systemic treatment of the patients, or islet pretreatment before transplantation to reduce their thrombogenicity. In this thesis, low molecular weight dextran sulfate (LMW-DS) is shown to reduce activation of the complement and coagulation systems and decrease the cell infiltration into the islets in vitro and in vivo, in both a xenogenic and an allogenic setting. Based on these results, LMW-DS is now in clinical trials.