Diabetes is a chronic metabolic disorder characterized by inappropriately elevated glucose levels as a result of impaired pancreatic β cell function and insulin resistance. Extensive studies have been conducted to elucidate the mechanism involved in the development of β cell failure and death under diabetic conditions such as hyperglycemia, hyperlipidemia, and inflammation. Of the plethora of proposed mechanisms, endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and oxidative stress have been shown to play a central role in promoting β cell dysfunction. It has become more evident in recent years that these 3 factors are closely interrelated and importantly aggravate each other. Oxidative stress in particular is of great interest to β cell health and survival as it has been shown that β cells exhibit lower antioxidative capacity. Therefore, this review will focus on discussing factors that contribute to the development of oxidative stress in pancreatic β cells and explore the downstream effects of oxidative stress on β cell function and health. Furthermore, antioxidative capacity of β cells to counteract these effects will be discussed along with new approaches focused on preserving β cells under oxidative conditions.

Following the success obtained with transplantation of fresh human islets under steroid-free immunosuppression, this trial evaluated the transplantation of islets that had undergone a period of in vitro culture and the potential of tumor necrosis factor (TNF-alpha) blockade to improve islet engraftment. Subjects included 16 patients with type 1 diabetes mellitus (T1DM); half were randomly assigned to receive Infliximab immediately preceding initial infusion. Immunosuppression consisted of daclizumab induction and sirolimus/tacrolimus maintenance. Out of 16 subjects 14 achieved insulin independence with one or two islet infusions; adverse events precluded completion in two. Without supplemental infusions, 11/14 (79%) subjects were insulin independent at 1 year, 6/14 (43%) at 18 months; these same subjects remain insulin independent at 33+/-6 months. While on immunosuppression, all patients maintained graft function. Out of 14 patients, 8 suffered chronic partial graft loss, likely immunological in nature, 5 of these received supplemental infusions. Currently, 11 subjects remain on immunosuppression, 8 (73%) are insulin independent, two with supplemental infusions. Insulin independent subjects demonstrated normalization of HbA1c, fructosamine and Mean Amplitude of Glycemic Excursions (MAGE) values. No clinical benefit of infliximab was identified. These results demonstrate that transplantation of cultured human islet allografts results in reproducible insulin independence in all subjects under this immunosuppressive regimen, comparable to that of freshly transplanted islets (Edmonton protocol).

OBJECTIVE: To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS: We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS: Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS: We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA-binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA-binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining β-cell viability. For these reasons we have developed novel methods for the specific assessment of β-cell content and viability in human islets based on cellular composition analysis through laser scanning cytometry (LSC) coupled with identification of β-cell-specific apoptosis at the mitochondrial level. Our novel analytical methods hold promise to prospectively analyze clinical islet transplantation preparations and predict functional performance, as suggested by the observed correlation with in vivo analysis of islet potency in immunodeficient rodents.