Immunol Res (2013) 57:210–221 DOI 10.1007/s12026-013-8437-4

IMMUNOLOGY & MICROBIOLOGY IN MIAMI

Transdisciplinary approach to restore pancreatic islet function Carmen Fotino • R. Damaris Molano Camillo Ricordi • Antonello Pileggi

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Published online: 15 November 2013 Ó Springer Science+Business Media New York 2013

Antonello Pileggi

Abstract The focus of our research is on islet immunobiology. We are exploring novel strategies that could be of assistance in the treatment and prevention of type 1 diabetes, as well as in the restoration of metabolic control via transplantation of insulin producing cells (i.e., islet cells). The multiple facets of diabetes and b-cell replacement encompass different complementary disciplines, such as immunology, cell biology, pharmacology, and bioengineering, among others. Through their interaction and integration, a transdisciplinary dimension is needed in order to address and overcome all aspects of the complex puzzle toward a successful clinical translation of a biological cure for diabetes. Keywords Islet transplantation  Autoimmunity  Rejection  Engraftment  Inflammation  Innate immunity  Bioengineering  Implantable device  Diabetes  Type 1 diabetes  Diabetes mellitus  Cell transplantation  Cellular therapies  Transplant microenvironment  Imaging  Hyperbaric oxygen therapy  ATP  Purinergic receptors  P2X7  CD39  CD73  Adenosine (Ado)  Beta cell  Immunotherapy  Metabolic control  Noninvasive imaging  Bioluminiscence  Confocal microscopy  Live imaging

The incidence of diabetes mellitus is growing at unprecedented rates in recent years, reaching the proportions of a global epidemic. Diabetes is one of the major causes of

C. Fotino  R. D. Molano  C. Ricordi  A. Pileggi (&) Cell Transplant Center, Diabetes Research Institute, University of Miami, Miami, FL, USA e-mail: [email protected] C. Ricordi  A. Pileggi DeWitt-Daughtry Department of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA C. Ricordi  A. Pileggi Department of Microbiology and Immunology, University of Miami Miller School of Medicine, Miami, FL, USA C. Ricordi  A. Pileggi Department of Biomedical Engineering, University of Miami, Miami, FL, USA C. Ricordi Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA

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chronic morbidities and mortality worldwide, with a great financial impact for diabetes management, including direct medical cost and indirect costs (i.e., disability, work loss, and premature mortality) [1]. Type 1 Diabetes Mellitus (T1DM) is a chronic autoimmune disorder resulting from genetic predisposition, immunological defects, and environmental factors concurring to the development of autoreactive T cells [1, 2] that mediate the destruction of insulin-producing pancreatic b-cells leading to insulinopenia and hyperglycemia requiring life-long dependence on exogenous insulin [3, 4]. It is estimated that T1DM currently may affect up to three million people in the USA alone, and it is predicted to rise in frequency. Moreover, the incidence of T1DM is spreading outside of the historically identified as at risk individuals and affecting also developed countries. To improve health and quality of life of people affected with diabetes, safe and effective methods to achieve and maintain normoglycemia are needed. Currently, these include the administration of exogenous insulin, diet, and exercise. Unfortunately, standard medical treatment cannot

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attain tight glycemic control in the majority of the patients. Unbalanced metabolic control results in the development of progressive, debilitating complications including cardiovascular diseases (heart disease, high blood pressure, and stroke), neuropathy (reduced sensation in extremities and gastric paresis), nephropathy (kidney failure requiring dialysis and kidney transplantation), retinopathy (leading to blindness), and varied biochemical imbalances, all of which negatively influence quality of life and life expectancy of people with diabetes [1, 2]. Intensive insulin therapy can reduce and prevent the progression of diabetic complications, but is associated with an increased frequency of severe hypoglycemia, at times life-threatening. Preservation of adequate and functional b-cell mass is a desirable goal toward the achievement of physiologic metabolic control in patients with diabetes. Desirable therapeutic regimens should be effective (alone or in combination), readily accessible, and without severe risks. Therapeutic options that have been proposed include the following (Fig. 1): (1) prevention studies in high-risk subjects; (2) early interventions at the time of diabetes onset; (3) delayed interventions to restore self-tolerance and b-cell regeneration; and (4) restoration of b-cell mass via islet transplantation (i.e., islets obtained from deceased donor human pancreas; xenogeneic sources, and in the future, stem-cell derived insulin-producing cells) [5]. Unfortunately, to date the success of prevention and intervention studies in humans has been disappointing, with only transient measurable effects of immunotherapy delaying the demise of b-cells [5]. In patients with hyperglycemia, restoration of b-cell mass can be attained via transplantation of allogeneic pancreatic islet cells (either isolated islets or whole pancreas transplantation) (Fig. 1), as demonstrated by the

steady clinical progress of the last three decades [6–8]. However, in order to prevent graft rejection and autoimmunity recurrence, lifelong immunosuppression is required [9, 10], which associates with untoward side effects (i.e., increased risk of opportunistic infections and neoplasms, as well as the development of progressive organ toxicity) [11– 20]. Thus, the risks associated with chronic immunosuppression currently limit the indication of islet transplantation to the most severe cases of T1DM in adults and preclude its application in juveniles. Furthermore, clinical islet transplantation is currently performed via embolization into the portal system, which leads to a substantial loss of functional islet mass due to the activation of nonspecific inflammation and hypoxia immediately after transplantation. The activation of innate immunity that occurs at the time of transplantation as a result of ‘danger’ signals (i.e., ischemia/reperfusion-like injury) may amplify the intensity of adaptive immune responses negatively affecting the fate of the graft. Thus, alternative sites of implant and novel protocols of immunomodulation are needed for islet transplantation to become a more widely applicable treatment of diabetes. The focus of our research is on islet immunobiology. We are exploring novel strategies that could be of assistance in the treatment and prevention of T1DM, as well as in the restoration of metabolic control via islet (or insulinproducing cell) transplantation. The multiple facets of diabetes and b-cell replacement encompass different complementary disciplines, such as immunology, cell biology, pharmacology, and bioengineering, among others. Through their interaction and integration, a transdisciplinary dimension is needed in order to address and overcome all aspects of the complex puzzle toward the successful clinical translation of a biological cure for diabetes.

Fig. 1 Schematic representation of b-cell mass during the progression of T1DM and after b-cell replacement. Subjects with genetic susceptibility may develop progressive loss of b-cell mass. Potential interventions in high-risk subjects or at the time of diabetes onset are aimed at preservation of b-cell mass (green arrow). After established

hyperglycemia, restoration of b-cell mass and function may be obtained by transplantation of insulin-producing cells (namely, islets) for which strategies are aimed at preservation of long-tern functional b-cell mass (red arrow)

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Exploring the immunomodulatory properties of hyperbaric oxygen therapy Hyperbaric oxygen therapy (HOT) is a safe and noninvasive therapy that has been clinically used to improve oxygen supply to hypoperfused tissues. HOT’s antiinflammatory properties [21–25] and ability to mobilize bone marrow stem cells [26–30] have been recognized and have been associated with its beneficial effect. In a murine Lupus model, HOT was associated with reduced mortality, decreased proteinuria, altered lymphocyte-subset redistribution, reduced anti-DNA antibody titers, and amelioration of immune-complex deposition [31]. HOT was also shown to improve islet-transplant engraftment in mice [32, 33]. We sought to evaluate the effects of HOT on the development of diabetes. The nonobese diabetic (NOD) mouse is considered a useful ‘preclinical’ model of T1DM. Similar to clinical T1DM, the insulin-producing cells in pancreatic islets are the target of a cell-mediated immune destruction, resulting in hyperglycemia. Several features (i.e., genetic predisposition, immune-cell defects, among others) of clinical T1DM have their correlation in NOD mice. Female NOD mice develop autoimmune diabetes spontaneously during their life span starting at approximately 12 weeks of age (*80 % incidence). Moreover, diabetes onset can be accelerated by the administration of cyclophosphamide (CyP) through putative depletion of regulatory T cells (Tregs) and development of hyperglycemia within 2 weeks. The NOD mouse model is useful to evaluate the efficacy of interventions aimed at: (1) the prevention of autoimmunity (high-risk subjects), (2) halting the progression of b-cell destruction (at the time of diabetes onset); and (3) restoration of b-cell function by islet transplantation (established diabetes). In our study, HOT resulted beneficial and reduced the incidence of diabetes onset in NOD mice [34]. In the accelerated onset model, 85.3 % of the control mice developed diabetes after CyP injection within 35 days, while animals receiving daily HOT starting the day before CyP administration developed diabetes only in 48 % of the cases. The effect of HOT appeared to be dependent on oxygen concentrations, with the highest effect obtained with 100 % O2 at 2.0 atmospheres absolute (ATA) and lower protection from diabetes when \100 % O2 was given, indicating that hyperbaric treatment per se was not sufficient and that rather high oxygen is required to elicit the protective effect of HOT in this model. In the model of spontaneous diabetes occurrence, HOT was started at 4 weeks of age (60-min daily sessions at 2.0 ATA) and resulted in delayed and reduced diabetes occurrence in female NOD mice from 85 % in controls to 65 % in the HOT experimental group [34]. Higher levels of cytoprotective cytokine interleukin (IL)-10 were detected in the HOT group compared to controls. The histopathological

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analysis of the pancreas in the prevention studies demonstrated reduced infiltrating cells (T cells and B cells) and better preservation of islet structure in the HOT group. We also evaluated the impact of 2-week HOT regimen in prediabetic female NOD mice (13-week old). Histopathological assessment revealed reduced insulitis, lower rates of bcell apoptosis, and a significant increase in b-cell proliferation, assessed by bromodeoxyuridine incorporation. This phenomenon was also observed in immunodeficient mice with the same genetic background (namely, NOD.scid) treated with HOT. However, the proportion of proliferating b-cells were higher in the islets of NOD mice with insulitis, possibly as a result of increased metabolic demand and activation of survival/replication pathways under stress conditions. In an attempt to elucidate the possible immunological mechanisms underlying the protected effect conferred by HOT, multiple parameters were assessed. A higher frequency of resting (CD4 ? CD62L?) and lower activated (CD4 ? CD69?) T cells were found in HOT-treated mice. In addition, splenocytes obtained from long-term euglycemic HOT-treated mice were able to suppress the transfer of the disease when co-injected with splenocytes obtained from recently diabetic NOD mice, indicating that cell populations present in HOT-treated mice were able to suppress autoreactive cells in this model. Furthermore, CD4? T cells and splenocytes obtained from mice receiving HOT showed lower proliferation rates than control in response to anti-CD3 antibody stimulation in vitro. Activation markers in antigenpresenting cells were also affected by HOT therapy, displaying lower expression of co-stimulation markers (CD40, CD80, CD86, and MHC-II) in CD11c? dendritic cells (DC), compared to controls. These data indicate reduced immune activation in HOT mice that may have contributed ultimately to the reduction of insulitis and protection from autoimmune diabetes progression in this model. We then investigated the potential effect of HOT therapy on the reversal of the established disease. Once hyperglycemia was detected, mice were started on insulin therapy (by the use of a subcutaneous insulin pellets) and the glucagon-like peptide (GLP)-1 receptor agonist exenatide (administered via subcutaneous osmotic pump) with or without daily HOT therapy. Addition of HOT to the exenatide treatment delayed the onset of diabetes, but the disease recurred in most of the animals, as in the control groups. Similarly, HOT treatment in spontaneously diabetic NOD mice receiving syngeneic islets (a model of acute diabetes recurrence) was insufficient to protect most of the mice from the recurrence of the disease. Collectively, these data seem to indicate that the explored therapies are insufficient to suppress an already activated and aggressive autoimmune process. Taking together, our studies indicate that HOT beneficial effects on b-cell mass preservation (namely, resulting

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from the cumulative effect of reduction of cell death and induction of proliferation) and reduction of immune-cell activation (Fig. 2) make HOT a safe valuable strategy that could be used to enhance and improve clinical therapies devised for diabetes prevention and treatment in the future. Current efforts in our laboratory aim at exploring potential synergy of HOT with immunotherapy to halt autoimmune diabetes progression at the time of onset in NOD mice.

Understanding the role of ATP and its signaling in islet immunity Innate immunity encompasses highly regulated, complex cellular, and humoral responses elicited as first-line of defense of our organism in response to insults. Any insult results in ‘danger’ signals aimed at ‘damage control’ while more specialized immune responses are mounting. After the resolution of the insult, the inflammation process subsides thanks to immune regulatory signals in the local microenvironment. The pivotal role of the activation of inflammation pathways in the maintenance and amplification of immune responses is associated with various medical conditions, including autoimmunity and organ transplant rejection. The activation of innate immunity generated at the time of islet transplantation as a result of ‘danger’ signals (i.e., ischemia/reperfusion-like injury) may amplify the intensity of adaptive immune responses negatively affecting the fate of the graft [35] via the modulation of molecular pathways associated with stress-activated protein kinases [36–42]. Extended periods of ischemia endured by vascularized grafts can increase the risk of primary nonfunction or delayed function that are associated with higher incidence of rejection episodes and shorten graft survival [43–46]. It is conceivable that

Fig. 2 Schematic representation of the observed effects of hyperbaric oxygen therapy (HOT) in NOD mice. APC antigen-presenting cells, Teff effector T lymphocyte

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nonspecific inflammation may also contribute to the reactivation of the autoimmune process, worsening graft outcome in the context of islet transplantation in patients with T1DM. Furthermore, the inflammation generated at the time of transplant may impair the efficacy of immunomodulation protocols aimed at inducing immune tolerance. Experimental and clinical data support the beneficial impact of peri-transplant anti-inflammatory treatment on the engraftment and function of islet grafts. We demonstrated that in vitro preconditioning with cytoprotective agents (i.e., to induce heme oxygenase-1, HO-1, upregulation) helps preventing islet cell death and also reducing immunogenicity (MHC class II expressing cells) [47], resulting in improved engraftment of a syngeneic marginal islet mass [48], prolongation of survival or even acceptance when transplanted into fully MHC-mismatched recipient mice without immunosuppression [47]. Improved allogeneic islet graft survival was also demonstrated after peri-transplant anti-inflammatory treatment, such as alpha-1 anti-trypsin [49]. Great synergy was reported using cytoprotective agents (i.e., caspase inhibitors) in combination with co-stimulation blockade with Cytotoxic T-Lymphocyte Antigen 4 (CTLA4)Ig in a murine islet allograft model [50]. A recent retrospective analysis of the Collaborative Islet Transplant Registry (CITR) has shown that sustained insulin independence in islet transplant recipients was higher when using tumor necrosis factor (TNF)-a blockade in the peri-transplant period [51]. Recent clinical trials have shown that prolonged use of anti-Lymphocyte Function-associated Antigen (LFA)-1 antibodies (i.e., Efalizumab) is associated with high rates of insulin independence after single-donor islet transplantation using anti-CD25 antibody induction with maintenance immunosuppression based on either molecular Target of Rapamycin (mTOR) inhibitors and calcineurin inhibitors (CNI)[52], or by induction with anti-CD25 treatment and lymphodepletion (thymoglobulin) followed by maintenance with de novo purine synthesis inhibitors (Mychophenolic acid), mTOR inhibitor [53, 54]. In the context of autoimmune diabetes models, LFA1 blockade using either anti-LFA-1 or anti-Intercellular Adhesion Molecule (ICAM)-1 antibodies alone or in combination effectively prevented both the onset and also the transfer of the disease in a murine model of T1DM, the NOD mouse [55–57]. The impact of a short-term anti-LFA-1 antibody treatment alone in spontaneously diabetic NOD mice was partially efficacious, resulting only in delayed allograft rejection [58]. Co-stimulation blockade can improve islet allograft survival in spontaneously diabetic NOD mice [59]. A synergistic effect has been achieved by combining anti-LFA-1 antibody treatment with co-stimulation blockade (i.e., CTLA4Ig or CD154 antibody), with T cell depletion or immunosuppression (i.e., mTOR inhibitors) in spontaneously diabetic NOD mice receiving allogeneic or xenogeneic islet grafts [58, 60]. Synergy of stimulation blockade was also

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observed in the challenging model of allogeneic islet transplantation into spontaneously diabetic NOD mice [61]. Our working hypothesis is that combinatorial treatments that dampen early inflammation in the peri-transplant period may enhance the success of otherwise partially effective tolerogenic protocols for allogeneic grafts. We have begun exploring the role of adenosine 50 -triphosphate (ATP) signaling in the context of islet immunobiology (Fig. 3). ATP is a small molecule considered as the primary source of energy of the cells. High concentrations of ATP (1–10 mM) are present in the intracellular space of every living cell, while in the extracellular space the concentrations of ATP are maintained at low levels (1–10 nM). Under physiologic conditions, ATP is secreted by exocytosis, but it increases rapidly in response to tissue stress and damage exerting its effect through the activation of the P2 purinergic receptors [62]. Two classes of P2 receptors have been characterized, P2XRs and P2YRs. P2X receptors are membrane-ion channels gated by extracellular ATP. Among them, P2X7R is the best characterized. P2Y receptors are G-protein-coupled receptors gated by adenosine diphosphate (ADP), uridine diphosphate (UDP), and uridine triphosphate (UTP). Adenosine 50 -tri-phosphate in the extracellular space is rapidly degraded by the plasma membrane ecto-nucleotidase CD39 into ADP/adenosine monophosphate (AMP) and then CD73 converts AMP into adenosine (Ado) [63]. CD39 expression is regulated by several cytokines, oxidative stress, and hypoxia through the transcription factor Sp1, Stat-3, and zinc finger protein growth factor independent-1 transcription factor (Gfi-1) [64]. CD73 is upregulated under hypoxic conditions, as well as by the presence of several pro-inflammatory

Fig. 3 Schematic representation of the putative role of ATP signaling in islet immunobiology. Ado adenosine, APC antigen-presenting cell, ATP adensosine-50 -triphosphate, ADP adenosine diphosphate, AMP adenosine monophosphate, b beta cell, EC endothelial cell, P2XR purinergic receptor, Teff effector T lymphocyte, Treg regulatory T lymphocyte

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mediators, such as transforming growth factor (TGF)-b, TNFa, and interleukin (IL)-1b [65, 66]. The activity of CD39 is reversible, while the activity of CD73 is virtually irreversible; thus, CD73 represents an important step in the conversion of pro-inflammatory ATP into Ado that is an important regulator of immune activation. It has been shown that Ado suppresses CD4 T cell activation and reduces CD25 expression after stimulation with anti-CD3 antibody [67]. Both CD39 and CD73 are surface markers of Foxp3 Treg cells. Their coexpression and the production of Ado determine the suppressive capabilities of Treg cells [68, 69]. Furthermore, CD73 is upregulated on Tregs after antigenic stimulation and the Ado derived acts through A2aRs (Adenosine A2a receptors) on T-effector (Teff) cells to downregulate NF-KB, which is linked to the inhibition of various cytokines and chemokines [70]. Extracellular Ado, derived from the activity of CD73 is regulated by adenosine deaminase (ADA), an enzyme that degrades adenosine to inosine. Adenosine deaminase is present at high levels in DCs from NOD mice. Indeed, DCs from ADA-deficient NOD mice fail to efficiently trigger autoimmune diabetes, suggesting the important role of ADA for DC-mediated T cell activation [71]. We have already demonstrated in a murine model of islet and heart allograft rejection the role of ATP and its signaling through P2XRs (specifically P2X7R) [72, 73]. We observed that modulation of P2XRs, in particular P2X1R and P2X7R, is induced in syngeneic and allogeneic grafts in vivo in the peri-transplant period. In our model of islets allotransplantation the treatment with oxidized ATP (oATP), a shiff-base molecule that antagonizes P2X7R, was able to promote long-term graft survival in 30 % of the recipients. This effect was also related with a reduction of CD4? Teff and Th17 cells that are important in promoting an alloimmune response; and a preservation of Tregs, which on the contrary promote graft acceptance [73]. We are currently exploring the role of ATP and its signaling in islet cells, and their involvement in islet cell directed immune responses during autoimmunity and rejection. It has been shown that INS-1e cells (derived from the parental rat insulinoma INS-1 cell line) express multiple purinergic receptors and that ATP reduces glucose-induced insulin secretion as a direct effect of inhibition through P2X and stimulation through P2Y receptors [73]. We have previously described the role of ATP in mediating a positive autocrine signal for insulin secretion in isolated human islets during metabolic challenge in vitro [74]. Our working hypothesis is that co-release of insulin and ATP may contribute to the amplification of inflammation through P2XR at the immune synapse between pancreatic islets and T cells. Moreover, a functional CD39 is expressed in human b-cells [75], and its inhibition (i.e., using ARL67156) results in increased basal insulin secretion from islets incubated at low glucose concentration, revealing that

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human islet cells release ATP and that endogenous ectoATPases are fully effective, possibly explaining why exogenously added CD39 did not reduce basal insulin secretion in low glucose [74]. Modulation of the ATP signaling may re-establish the immunobiological basis for tolerance toward autoantigens, thus providing the proof-of-principle to develop novel therapeutic to target autoimmune diabetes, as well as contribute to the modulation of alloresponses in islet cell transplants. Our current efforts aim at characterizing the presence of molecules (potentially targetable) related to the pathways on islet and immune cells that help breaking down ATP to increase powerful pro-tolerogenic byproducts. Very encouraging ongoing studies suggest great synergy of the blockade of the ATP/purinergic receptor signaling when used with co-stimulation blockade in experimental models of islet transplantation. Our approach could be synergistic with other immunotherapies aimed at protecting islet cells at the time of autoimmune diabetes onset as well as in the transplant setting.

Re-engineering the transplant microenvironment Transplantation of pancreatic islets represents a clinical therapeutic option to preserve and/or restore b-cell function in patients with diabetes [8, 76]. Since the 1970s, islets are embolized into the hepatic portal system by a minimal-invasive technique consisting of transhepatic cannulation of the portal vein under ultrasound and fluoroscopy guidance followed by sealing of the tract with thrombostatic treatment. Alternatively, in patients at risk of bleeding, the transplant is performed by cannulation of a tributary of the portal vein using open surgery (mini-laparotomy) or laparoscopic approach. In recent years, the need for alternative extra-hepatic sites for islet implantation has been recognized, prompting

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active research for the most suitable microenvironment to promote engraftment, minimize early inflammation and islet cell death, while achieving sustained function [77, 78]. Extra-hepatic sites tested in experimental animals include the subcapsular kidney space [79], spleen [78], intraabdominal vascularized devices [80, 81], subcutaneous space [82], omental pouch [83–90], intramuscular [91, 92], the eye [93–95], excluded venous segment [96] and intestinal segments [96, 97], and bone marrow [98], among others [99]. Extra-hepatic sites already tested in humans include muscle [100–103], peritoneal cavity (to accommodate large microencapsulated islets) [104, 105], and the bone marrow [106]. Emerging bioengineering approaches are opening new opportunities to enhance islet engraftment and survival [107, 108]. One of the strategies we are exploring is the modification of the transplant microenvironment to favor long-term function of islet grafts. Indeed, while the current approach to prevent rejection is based on the administration of high-dose systemic immunosupression, we believe that a more targeted intervention that spares the recipient from the deleterious side effects of these drugs would be a key advancement in the field. Our approach is based on the combination of either short-term or low-dose administration of immunosuppressive drugs with the administration of anti-inflammatory agents. This can be achieved by either systemic or local delivery of combinations of selected compounds and will bear the advantage of efficacy with much reduced side effects. We have been working on the optimization of an alternative site for islet implantation that would also allow the delivery of immunomodulating drugs directly to the graft site. This could dramatically reduce the doses of drugs needed, compared with systemic administration of the medication. We have already demonstrated the long-term function of syngeneic pancreatic islets into chemically induced diabetic

Fig. 4 Schematic of the biohybrid device implantation and islet transplant

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Lewis rats using a biohybrid device consisting of a cylindrical mesh (made of stainless steel or with the biocompatible synthetic polymer polyetheretherketone, PEEK) [82]. The device was implanted subcutaneously 6 weeks prior to the transplant to allow for vascularization (Fig. 4). Transplantation of syngeneic islets in the metal devices resulted in reversal of diabetes in seven of the eight recipients with a median time of 6 days. Islets are highly vascularized clusters receiving *10 % of pancreatic blood flow in their native environment despite constituting only *1–2 % of the gland. Poor oxygen diffusion to the islets after transplantation may impair cell viability and performance [109–112]. Creating a pre-vascularized implant microenvironment should enhance islet cell survival into biohybrid devices, increase local oxygen supply, and accelerate islet neovascularization after transplant. This approach provides adequate mechanical protection and sustained graft function, while facilitating implantation, replenishing, biopsy, or retrieval as needed [82]. An important characteristic of biohybrid devices is the confinement of transplanted tissue within the device, which makes it possible to deliver localized treatments. We have engineered a biohybrid device with an infusion port for local treatment [113–115], which can provide sufficient protection against rejection and allow for long-time function using much smaller doses than in a corresponding systemic administration (Fig. 4). Thus, the serious side

Fig. 5 Noninvasive, live imaging for islet immunobiology

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effects associated with long-term systemic immunosuppression (i.e., increased susceptibility to opportunistic infections and organ toxicity, among others) could be avoidable. Preliminary studies ongoing in our laboratory are evaluating the impact of systemic and local immunotherapy on islet allograft survival [115]. We aim at developing clinically relevant combinatorial treatments that could support long-term islet allograft function using reduced immunosuppression needs.

Noninvasive live imaging to study islet immunobiology The complexity of biologic phenomena is the result of interactions between cells and the microenvironment that are difficult to fully reproduce in vitro. The availability of in vivo experimental models, such as genetically modified rodents has been instrumental in the understanding of biologic systems. Moreover, the rapidly evolving field of noninvasive live imaging is creating new exciting opportunities for islet immunobiology. There is a number of existing technologies available to the research community [116], including magnetic resonance imaging MRI [117, 118], positron emission tomography (PET) and radionucleotide scintigraphy (SCI) [119, 120], bioluminescence imaging (BLI) [121], confocal/multiphoton fluorescent microscopy (CFM/MPM) [93, 94, 122–128], among others.

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Fig. 6 Bioluminescence imaging of islet grafts. a Islets obtained from FVB-luc-GFP mice (FVB-Tg(CAG-luc,-GFP)L2G85Chco/J) were imaged before transplantation, 5 min after addition of luciferin to the culture media. b Diabetic C57Bl/6 mice were transplanted with islets obtained from FVB-luc-GFP mice either under the kidney capsule (KC) or under a pre-vascularized subcutaneous device

(DEVICE) and were imaged using a charge-coupled device (CCD caliper life sciences, Hopkinton, MA, USA) at different time points after transplantation until rejection. Signal disappearance coincided with reappearance of hyperglycemia. Mice were injected with Luciferin (150 mg/kg IP) 25–35 min before imaging session

Each approach bears some pros and cons. While some may also have an immediate clinical applicability, others represent invaluable research tools to study different aspects of islet biology. We use different noninvasive live imaging approaches applied to islet immunobiology, namely CFM/MPM and BLI (Fig. 5). We have evaluated the feasibility of studying islet immunity by noninvasive imaging techniques using the anterior chamber of the eye as a site for islet implantation in mice [94]. In a model of fully MHC-mismatched islet transplantation, we have established that islets are rejected and have evaluated longitudinally the dynamics of immune-cell infiltration and islet destruction. Recently, we have studied the progression of autoimmune diabetes implanting syngeneic islets in NOD.scid mice that were the reconstituted with splenocytes from recently diabetic NOD mice. The use of fluorescently labeled immune-cell populations allows us to follow and study the dynamic pattern of the immune cells as autoimmunity progress in vivo. Collectively, the model enabled (1) longitudinal, noninvasive live imaging of transplanted tissues; (2) in vivo cytolabeling to assess cellular phenotype and viability in situ; (3) local intervention by topical application or intraocular injection; (4) real time tracking of infiltrating immune cells in the target tissue; and (5) cellular resolution. Limitations of this technique include the fact that it is not the native environment, and the limited access to the transplant area without providing information on other concomitant events occurring elsewhere (i.e., lymph nodes). We also are using BLI to monitor the fate of islet grafts or adjuvant cells that express luciferase, which emits light

when the substrate luciferin is administered to the animals. For instance, we have performed fully MHC-mismatched islet transplant either under the kidney capsule or into a subcutaneous pre-vascularized device. The transgenic expression of Luc allowed monitoring the rejection detected as lost of light intensity overtime (Fig. 6). This approach allows us to monitor the faith of cell inocula (i.e., distribution, persistence, homing etc.) in the all animal longitudinally. Limitations of this approach are the lack of cellular resolution and interference of the surrounding tissue to the light emission that can be detected.

Conclusions The multiple facets of diabetes and b-cell replacement require a transdisciplinary team approach in order to achieve successful translation to the clinic. Our sequential, integrated approach aims at combining multiple interventions and approaches towards getting closer to the ultimate goal of a biological cure for diabetes. Acknowledgments We are grateful to the faculty and staff at the Diabetes Research Institute (www.DiabetesResearch.org) and to the collaborators from multiple national and international institutions, including the members of the Diabetes Research Institute Federation (/ www.diabetesresearch.org/DRI-Federation-Members?) and of The Cure Alliance (www.TheCureAlliance.org) contributing to our transdisciplinary research team endeavors. The work discussed herein is integral part of the Diabetes Research Institute’s BioHubÓ Mini Organ Program. Invaluable assistance was obtained through the Diabetes Research Institute’s Cores (Preclinical Cell Processing &

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218 Translational Models, Human cGMP Cell Processing, Imaging, Flow Cytometry, Histology, and Administrative) and through the University of Miami Division of Veterinary Resources and the DNA Core for Bioluminiscence Imaging. Procedures involving animals were performed under protocols approved and monitored by the University of Miami IACUC under an Animal Welfare Assurance on file (A-322401, effective 12/4/02) with the Office of Laboratory Animal Welfare (OLAW), National Institutes of Health, and full accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care. The biohybrid device was designed and manufactured by Biorep, Inc. (Miami, FL). Human islets were obtained through NIHNCRR Islet Cell Resources (ICR) and subsequently from the Integrated Islet Distribution Program (IIDP). This work was supported in part by grants from the American Diabetes Association (7-13-IN-32; to A.P.), the Juvenile Diabetes Research Foundation International (172012-361, 17-2010-5, 4-2008-811, 4-2004-361, to C.R. and A.P.), The Leona M. and Harry B. Helmsley Charitable Trust, the National Institutes of Health (5U19AI050864-10 to A.P.; U01DK089538 to A.P.; 5U42RR016603-08S1 to C.R.; 1U01DK70460-02 to C.R.; 5R01DK25802-24 to C.R.; 5R01DK56953-05 to C.R.), the University of Miami Interdisciplinary Research Development Initiative (A.P.), and the Diabetes Research Institute Foundation (to A.P., C.R.), and Converge Biotech (A.P. and C.R.). Notably, the funding agencies had no role in the design and conduct of the study, collection, management, analysis and interpretation of the data, content, presentation, decision to publish, or preparation of the manuscript. Conflict of interest C.F., R.D.M., C.R. and A.P. own intellectual property that may be related to the topic discussed in this article. A.P. and C.R. are members of the scientific advisory board; and A.P., R.D.M., and C.R. are stock option holders in Converge Biotech, licensee of some of the intellectual property that may be related to the topic discussed in this article.

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