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-B (NF-B) 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-B؊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-B 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.

A lthough it is currently very clear that ␤-cells can replicate, albeit slowly, in vitro and in vivo, both basally and in response to a variety of maneuvers and stimuli (rev. in 1-3), the key components of the cell cycle machinery in the ␤-cell and the factors that regulate them are poorly understood at a molecular level. The retinoblastoma protein (pRb) (rev. in 4 -6) is a key gap-1/synthesis phase (G 1 /S) checkpoint gatekeeper. In its dephosphorylated (or active) state, pRb binds to the E2F family of cell cycle regulatory genes and leads to the transcriptional repression of downstream genes, with resultant cell cycle arrest. Conversely, when pRb is phosphorylated to form ppRb, it becomes inactive and releases E2Fs, removing transcriptional repression of critical cell cycle genes, and the cell cycle progresses. pRb can be phosphorylated by a number of kinases. These include cyclin-dependent kinase (cdk)-4 and -6, which form complexes with the D cyclins, as well as cyclin E and cdk-2, which also form a complex. Because of their importance, these cyclins and cdks are under tight regulatory control themselves. This regulation is principally inhibitory and is accomplished by inhibitory kinases or cyclin inhibitor proteins such as p16, p18, p21, p27, p53, p57, and others.The majority of the information described above on pRb and ppRb has been obtained in human and animal cancers and fibroblasts (4 -6). Surprisingly, given the current attention on ␤-cell replication, little is known regarding molecular control of the cell cycle in the ␤-cell. For example, we are unaware of any study examining either the presence of, or the phosphorylation status of, pRb in human or animal ␤-cells. On the other hand, there are some data that point to this pathway as being critical to the control of the cell cycle in ␤-cells. For example, SV-40 large T-antigen (TAg) is a transforming viral protein that interacts with p53 and pRb. TAg has been overexpressed in the ␤-cell of transgenic "RIP-TAg" mice and in cultured ␤-cell lines by Hanahan (7) and , and increased ␤-cell replication resulted. Ultimately, autonomous ␤-cell tumors develop in RIP-TAg mice.Homozygous disruption of the pRb gene in mice results in embryonic lethality (10). Heterozygous deletion results in adult animals characterized by the development of

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 SODtreated 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.

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 posttransplantation, islet grafts were retrieved for the determination of insulin content and vascular density. Compared with mocktreated 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 ␤-cell function in transplants. Diabetes 55:2429 -2436, 2006 T he Edmonton protocol for islet transplantation depends on the infusion of ϳ10,000 IE (islet equivalents)/kg body wt, requiring multiple cadaver pancreata per diabetic recipient (1-4). Despite the implantation of such a large quantity of islets, Ͻ30% of transplanted islets can survive the procedure and gain stable engraftment, and yet the mechanism underlying the loss of a vast majority of islet mass in the early posttransplantation phase remains elusive (4,5). Unlike whole-organ transplantation, by which grafts are implanted as vascularized tissue, islets are transplanted as single islets or islet clusters that are considered avascular following collagenase digestion and isolation. Although residual endothelial cells in isolated islets may contribute to islet revascularization (6,7), adequate intraislet blood flow requires the formation of a functional microvascular network that links engrafted islets to surrounding tissues. These data suggest that microvascular perfusion to newly transplanted islets does not resume immediately after transplantation and can take up to 2 weeks before the reestablishment of a functional microvasculature in islet grafts (8,9). This delay in islet revascularization can potentially deprive islets of oxygen and nutrients, resulting in islet cell death, particularly within the core of engrafted islets. There is mounting evidence that impaired islet revascularization is an independent factor that limits the success rate...