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Type 1 diabetes (T1D) is a major health problem throughout the world. In the U.S., it is estimated that about 1.5 million people suffer from T1D. Even when well controlled--by [...]

Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-α) as well as various chemotactic proteins, such as IFN-γ-induced protein 10, macrophage inflammatory protein (MIP)-Iα, MIP-1β, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet-inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis. Diabetes 61:2030-2036, 2012, Type 1 diabetes is a chronic inflammatory disorder of which the etiology is not fully understood. It is known, however, that both genetic and environmental factors are involved. Enteroviruses, particularly [...]

OBJECTIVE--Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant. RESEARCH DESIGN AND METHODS--The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-repeffusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation. RESULTS--Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controis. Diabetic routine recipients of catalytic antioxidant-treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection. CONCLUSIONS--Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation. Diabetes 59:1731-1738, 2010, Hypoxia is the leading cause of β-cell death during islet isolation and transplantation (1), with the highest percentage of islet graft loss and dysfunction occurring just days after transplantation (2,3). [...]

OBJECTIVE--Type 1 diabetes is a chronic endocrine disorder in which enteroviruses, such as coxsackie B viruses and echoviruses, are possible environmental factors that can trigger or accelerate disease. The development or acceleration of type 1 diabetes depends on the balance between autoreactive effector T-cells and regulatory T-cells. This balance is particularly influenced by dendritic cells (DCs). The goal of this study was to investigate the interaction between enterovirus-infected human pancreatic islets and human DCs. RESEARCH DESIGN AND METHODS--In vitro phagocytosis of human or porcine primary islets or Min6 mouse insuloma cells by DCs was investigated by flow cytometry and confocal analysis. Subsequent innate DC responses were monitored by quantitative PCR and Western blotting of interferon-stimulated genes (ISGs). RESULTS--In this study, we show that both mock- and coxsackievirus B3 (CVB3)-infected human and porcine pancreatic islets were efficiently phagocytosed by human monocyte-derived DCs. Phagocytosis of CVB3-infected, but not mock-infected, human and porcine islets resulted in induction of ISGs in DCs, including the retinoic acid-inducible gene (RIG)-I-like helicases (RLHs), RIG-I, and melanoma differentiation-associated gene 5 (Mda5). Studies with murine Min6 insuloma cells, which were also efficiently phagocytosed, revealed that increased ISG expression in DCs upon encountering CVB-infected cells resulted in an antiviral state that protected DCs from subsequent enterovirus infection. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification, and were type I interferon dependent. CONCLUSIONS--Human DCs can phagocytose enterovirus-infected pancreatic cells and subsequently induce innate antiviral responses, such as induction of RLHs. These responses may have important consequences for immune homeostasis in vivo and may play a role in the etiology of type 1 diabetes. Diabetes 59: 1182-1191, 2010, Type 1 diabetes, or insulin-dependent diabetes, is a chronic endocrine disorder characterized by the progressive loss of insulin-producing β-cells. In the majority of cases, type 1 diabetes is associated with [...]

OBJECTIVE--To describe the ability of nonhuman primate endocrine pancreata to reestablish endogenous insulin production after chemical β-cell destruction. RESEARCH DESIGN AND METHODS--Eleven monkeys (Macaca fascicularis) were rendered diabetic with streptozotocin. Eight diabetic monkeys received intraportal porcine islet transplantation. RESULTS--Two monkeys transplanted after 75 days of type 1 diabetes showed recovery of endogenous C-peptide production a few weeks after transplantation, concomitant with graft failure. Histological analysis of the pancreas of these monkeys showed insulin-positive cells, single or in small aggregates, scattered in the pancreas and adjacent to ducts. Interestingly, numerous [CK19.sup.+] cells costained with proinsulin and PDX-1 antibodies. Furthermore, the peculiar double phenotype glucagon-positive/ [GLUT2.sup.+] was observed. In these monkeys as well as in all others, the original islets showed no insulin staining. CONCLUSIONS--Our data provide evidence that, in nonhuman primates, the pancreas can reestablish endogenous insulin production after chemical β-cell destruction. This seems to be a nongeneralizable event with only 2 out of 11 monkeys recovering β-cell function. In these two monkeys, younger age and islet graft behavior might have played a role in triggering endogenous β-cell recovery., Until a few years ago, lesions of the endocrine pancreas, as occur in type 1 diabetes, were thought to be permanent and irreversible, since diabetic patients require hormone replacement therapy [...]

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a small fraction of implanted islets become engrafted. The underlying mechanisms remain elusive. To probe [...]

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a fraction of transplanted islets can survive and become engrafted, and yet the underlying mechanism remains unclear. In this study, we examined the effect of sirolimus, a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function. To distinguish the effect of sirolimus on immune rejection from its effect on islet engraftment, we used a syngeneic model. Diabetic mice were transplanted with 250 islets under the renal capsule, followed by treatment with sirolimus or vehicle for 14 days. Thirty days post-transplantation, islet grafts were retrieved for the determination of insulin content and vascular density. Compared with mock-treated controls, diabetic recipient mice receiving sirolimus exhibited impaired blood glucose profiles and reduced glucose-stimulated insulin secretion, correlating with reduced intragraft insulin content and decreased vascular density. Islets exposed to sirolimus for 24 h in culture displayed significantly diminished glucose-stimulated insulin release, coinciding with decreased pancreas duodenum homeobox-1 and GLUT2 expression in cultured islets. Furthermore, sirolimus-treated diabetic recipient mice, as opposed to mock-treated controls, were associated with dyslipidemia. These data suggest that sirolimus, administered in the early posttransplantation phase, is a confounding factor for reduced islet engraftment and impaired β-cell function in transplants., The Edmonton protocol for islet transplantation depends on the infusion of ~10,000 IE (islet equivalents)/kg body wt, requiring multiple cadaver pancreata per diabetic recipient (1-4). Despite the implantation of such [...]

Diabetes is a severe chronic disease that affects ~200 million individuals worldwide, with extremely debilitating effects and considerably high health care costs. The two major classes of diabetes, known as type 1 (previously known as insulin-dependent or juvenile-onset diabetes) and type 2 (non-insulin-dependent diabetes), share common symptoms such as hyperglycemia and the development of long-term complications, but they differ in many aspects, including their etiopathogenesis. New insights suggest that overlapping factors, formerly considered typical hallmarks of each specific type, can coexist in the same diabetic patient, making it difficult to support a sharp distinction between the two classes and, more importantly, to adopt appropriate therapeutic solutions. In type 1 and type 2 diabetic subjects, but even more in patients with combined types, multiple genetic factors play a role in determining susceptibility or resistance to the disease, and perhaps also the time of onset, the severity of the symptoms, the possibility of developing complications and, ultimately, the response to therapy. In this review, the therapeutic treatments currently under investigation, as well as the curative strategies envisioned for future applications, are reanalyzed considering the multifaceted and complex aspects of a continuum that can be just defined as 'diabetes.', The number of people affected by diabetes is dramatically rising in the modern societies on our planet (1). Several factors contribute to this higher incidence, including improved nutritional availability and [...]

Akt is an important intracellular mediator of [beta]-cell growth and survival in rodents. However, whether constitutive activation of Akt in human [beta]-cells enhances the survival and function of transplanted islets is unknown. In the current study, we examined the efficacy of constitutive activation of Akt in improving human islet transplant outcomes using a marginal mass model in diabetic severe combined immunodeficient (SCID) mice. Human islets transduced with adenoviruses encoding constitutively active Akt1 (Adv-CA-Akt) displayed increased total and phosphorylated Akt and Akt kinase activity compared with control islets. Expression of CA-Akt in human islets induced a significant increase in [beta]-cell replication and a significant decrease in [beta]-cell death induced by serum and glucose deprivation or chronic hyperglycemia. Two control groups of islets (1,500 uninfected or adenovirus LacZ [Adv-LacZ]-transduced human islet equivalents [IEQs]) transplanted under the kidney capsule of streptozotocin-induced diabetic SCID mice were insufficient to correct hyperglycemia. Importantly and in marked contrast to these controls, 1,500 Adv-CA-Akt-transduced IEQs were capable of restoring euglycemia in diabetic SCID mice. Moreover, blood glucose normalization persisted for at least 6 months. Human plasma insulin at day 54 after transplant was 10-fold higher in Adv-CA-Akt islet recipients (2.4 [+ or -] 0.4 ng/ml) compared with those receiving Adv-LacZ islets (0.25 [+ or -] 0.08 ng/ml) (P < 0.05). In summary, expression of CA-Akt in human islets improves islet transplant outcomes in a subcapsular renal graft model in SCID mice. Akt is an attractive target for future strategies aimed at reducing the number of islets required for successful islet transplantation in humans., Recent studies have demonstrated that human islet allograft transplantation can be a successful therapeutic option in the treatment of patients with type 1 diabetes (1-3). However, despite this enormous advance, [...]

The process of human islet isolation triggers a cascade of stressful events in the islets of Langerhans involving activation of apoptosis and necrosis and the production of proinflammatory molecules that negatively influence islet yield and function and may produce detrimental effects after islet transplantation. In this study, we showed that activation of nuclear factor-κB (NF-κB) and poly(ADP-ribose) polymerase (PARP), two of the major pathways responsible for cellular responses to stress, already occurs in pancreatic cells during the isolation procedure. NF-κB-dependent reactions, such as production and release of interleukin-6 and -8 and macrophage chemoattractant protein 1, were observed days after the isolation procedure in isolated purified islets. Under culture conditions specially designed to mimic isolation stress, islet proinflammatory responses were even more pronounced and correlated with higher islet cell loss and impaired secretory function. Here we present novel evidence that early interventions aimed at reducing oxidative stress of pancreatic cells and islets through the use of the catalytic antioxidant probe AEOL10150 (manganese [III] 5,10,15,20-tetrakis [1,3,-diethyl-2imidazoyl] manganese-porphyrin pentachloride [TDE-2,5-IP]) effectively reduces NF-κB binding to DNA, the release of cytokines and chemokines, and PARP activation in islet cells, resulting in higher survival and better insulin release. These findings support the concept that the isolation process predisposes islets to subsequent damage and functional impairment. Blocking oxidative stress can be beneficial in reducing islet vulnerability and can potentially have a significant impact on transplantation outcome., To become more successful, allotransplantation of human islets requires the availability of less toxic antirejection protocols and larger quantities of usable islets (1). In addition, the islet isolation process remains [...]

The major regulator of the gap-1/synthesis phase ([G.sub.1]/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of β-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in β-cells. Here, adenoviruses expressing cdk-4 and cyclin [D.sub.1] were used to explore rat and human pRb phosphorylation and β-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin [D.sub.1]/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin [D.sub.1] caused a threefold increase in [³H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human β-cell replication rates were increased 10-fold. Cdk-4 or cyclin [D.sub.1] overexpression did not adversely effect β-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin [D.sub.1]/cdk-4 for the induction of human and rodent β-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in β-cells, β-cell lines, and stem cell-derived β-cells., Although it is currently very clear that β-cells can replicate, albeit slowly, in vitro and in vivo, both basally and in response to a variety of maneuvers and stimuli (rev. [...]

The most commonly used technical approach to isolate human pancreatic islets intended for allotransplants generates a product that is hampered by mechanical and chemical insults, which dramatically reduce the mass of viable and functional transplantable cells. We tested a novel class of antioxidant chemical compounds (SOD mimics: AEOL10113 and AEOL10150) to protect human islets from oxidative stress in order to improve the preservation of the isolated tissue. Addition of SOD mimic in culture, after isolation, allowed for the survival of a significantly higher islet cell mass. Functional behavior and phenotypic cell characteristics of the SOD-treated islet preparations were preserved, as was the capacity to normalize diabetic mice, even when a marginal mass of islets was transplanted. The addition of SOD mimic during isolation, before culture, further reduced early cell loss. These results indicate that prompt interventions aimed at blocking oxidative stress can improve human islet survival, preserving a functional islet mass two- to threefold larger than the one usually obtained without adding any antioxidant compound. The ability to preserve functional islets without a dramatic loss represents a major advantage considering the scarce availability of islet tissue for clinical transplantation. Diabetes 51:2561-2567, 2002, In children, the whole pancreas transplant cannot be used, due to technical negative consequences, whereas transplantation of pancreatic islets has long been considered a potential curative treatment for type 1 [...]

Early impairment of islet function and graft loss limit the success of allogeneic islet transplantation. Non-specific inflammatory events occurring at the transplant site immediately after grafting, involving the production of [...]

Enzymatic digestion of donor pancreases is a vital step in human and large mammalian islet isolation. The variable enzymatic activities of different batches of commercially available collagenase is a major [...]

Typically, insulin release assays are the gold standard to determine the ability of isolated animal and human islets to respond to glucose concentrations with adequate insulin secretion. In vitro insulin release is measured using static and dynamic incubation techniques. The ability of islet grafts to correct hyperglycemia in streptozotocin-induced diabetic immunodeficient mouse recipients also provides solid information on insulin secretion, but it involves additional engraftment factors. Overall, these procedures are time-consuming. New alternative methodologies are now available to gain information on the secretory ability of isolated human islets. Adenoviral delivery of green fluorescent protein and timer protein in the C-peptide coding region of the insulin gene allows effective expression of fluorescent pro-insulin in the granules of rodent and human islet [beta]-cells. Incubation of human islets expressing ins-C-GFP and ins-C-Timer inserts, with high glucose or exposure to cytokines, induces differences in color and fluorescence intensity between treated and untreated control islets, easily appreciated by real-time microscopic analysis. The results correlate with data accrued using more classic methodologies. This new technical approach helps to better explore the activity of insulin release in isolated human islets and may contribute to identifying markers associated with islet functionality and relevant to islet performance after transplantation., Rita Bottino, (1) A.N. Balamurugan, (1,2) Xuehui Geng, (3) Peter Drain, (3) Suzanne Bertera, (1) Massimo Trucco (1) From the (1) Department of Pediatrics, Children's Hospital of Pittsburgh, Pittsburgh, Pennsylvania; [...]