Alberto Pugliese

Islet Transplantation in Type 1 Diabetes Mellitus Using Cultured Islets and Steroid-Free Immunosuppression: Miami Experience

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‐α) 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).

HLA-DQB1*0602 Is Associated With Dominant Protection From Diabetes Even Among Islet Cell Antibody–Positive First-Degree Relatives of Patients with IDDM

HLA-DQB1 alleles confer susceptibility and resistance to insulin-dependent diabetes mellitus (IDDM). We investigated whether the susceptibility alleles DQB1*0302 and DQB1*0201 affect progression to diabetes among islet cell antibody–positive (ICA+) first-degree relatives of IDDM patients and whether the protective allele DQB1*0602 can be found and is still protective among such relatives. We human leukocyte antigen–typed and periodically tested β-cell function (first-phase insulin release [FPIR] during the intravenous glucose tolerance test) in 72 ICA+ relatives, of whom 30 became diabetic on follow-up (longest follow-up 12 years); 54 (75%) relatives carried DQB1*0302 and/or DQB1*0201. The frequency of DQB1*0302 and DQB1*0201 and of the high-risk genotype DQB1*0302/DQB1*0201 did not differ significantly between diabetic relatives and those remaining nondiabetic. On follow-up, progression to IDDM was not statistically different for relatives with or without the DQB1*0302/DQB1*0201 genotype. However, those relatives with the DQB1*0302/DQB1*0201 genotype had a tendency to develop diabetes at an earlier age (log-rank P = 0.02). We found DQB1*0602 in 8 of 72 (11.1%) ICA+ relatives. Relatives with DQB1*0602 did not develop diabetes or show any decline of FPIR versus 28 of 64 DQB1*0602– relatives who developed IDDM (log-rank P = 0.006; Wilcoxons P = 0.02). The protective allele DQB1*0602 is found in ICA+ relatives who have minimal risk of progression to IDDM. Therefore, DQB1*0602 is associated with protection from IDDM both in population studies and among relatives with evidence of autoimmunity who should not enter prevention trials.

Self-antigen–presenting cells expressing diabetes-associated autoantigens exist in both thymus and peripheral lymphoid organs

Recent reports indicate that genes with tissue-restricted expression, including those encoding the type 1 diabetes autoantigens insulin, glutamic acid decarboxylase (GAD), and the tyrosine-phosphatase-like protein IA-2 (or ICA512), are transcribed in the thymus. The reported modulation of diabetes susceptibility by genetically determined differences in thymic insulin levels and studies in transgenic mice provide correlative and functional evidence that thymic expression of peripheral proteins is crucial for immunological self-tolerance. However, there are no specific data about the existence, tissue distribution, phenotype, and function of those cells that express insulin and other self-antigens in the human thymus. We find that the human thymus harbors specialized cells synthesizing (pro)insulin, GAD, and IA-2, mainly localized in the medulla, and we demonstrate such cells also in peripheral lymphoid organs (spleen and lymph nodes). Phenotypic analysis qualifies these cells as antigen-presenting cells (APCs), including both dendritic cells and macrophages. These cells often appear surrounded by apoptotic lymphocytes, both in thymus and spleen, and may therefore be involved in the deletion of autoreactive lymphocytes. Our findings demonstrate the existence of, and define the tissue distribution and phenotype of, a novel subset of APCs expressing self-antigens in human lymphoid organs that appear to be involved in the regulation of self-tolerance throughout life.

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells

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.

The diagnosis of insulitis in human type 1 diabetes

To the Editor: During a workshop concerning the histopathological characteristics of insulitis in human type 1 diabetes (fifth annual meeting of the JDRF Network for Pancreatic Organ Donors with Diabetes [nPOD], 10 February 2013, Jacksonville, FL, USA), a consensus opinion was reached on the criteria necessary for the diagnosis of insulitis, and a definition of insulitis was agreed, as detailed in the text box. Workshop attendees included many leading researchers in the

Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and β-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb+, n = 18) and without islet autoantibodies (AAb−, n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb+ donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = −0.58, P = 0.01) but not with age at disease onset. Residual β-cells were observed in all type 1 diabetes donors with insulitis, while β-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin+ islets compared with 2% of insulin− islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45+, CD3+, CD4+, CD8+, and CD20+ cell numbers within the insulitis (r = 0.53–0.73, P = 0.004–0.04), but not CD68+ or CD11c+ cells. The presence of β-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.

Network for Pancreatic Organ Donors with Diabetes (nPOD): developing a tissue biobank for type 1 diabetes.

The Network for Pancreatic Organ Donors with Diabetes (nPOD) was established to recover and characterize pancreata and related organs from cadaveric organ donors with various risk levels for type 1 diabetes (T1D). These biospecimens are available to investigators for collaborative studies aimed at addressing questions related to T1D natural history and pathogenesis.

Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients

Most autoimmune diseases (AID) are linked to an imbalance between autoreactive effector T cells (Teffs) and regulatory T cells (Tregs). While blocking Teffs with immunosuppression has long been the only therapeutic option, activating/expanding Tregs may achieve the same objective without the toxicity of immunosuppression. We showed that low-dose interleukin-2 (ld-IL-2) safely expands/activates Tregs in patients with AID, such HCV-induced vasculitis and Type 1 Diabetes (T1D). Here we analyzed the kinetics and dose-relationship of IL-2 effects on immune responses in T1D patients. Ld-IL-2 therapy induced a dose-dependent increase in CD4(+)Foxp3(+) and CD8(+)Foxp3(+) Treg numbers and proportions, the duration of which was markedly dose-dependent. Tregs expressed enhanced levels of activation markers, including CD25, GITR, CTLA-4 and basal pSTAT5, and retained a 20-fold higher sensitivity to IL-2 than Teff and NK cells. Plasma levels of regulatory cytokines were increased in a dose-dependent manner, while cytokines linked to Teff and Th17 inflammatory cells were mostly unchanged. Global transcriptome analyses showed a dose-dependent decrease in immune response signatures. At the highest dose, Teff responses against beta-cell antigens were suppressed in all 4 patients tested. These results inform of broader changes induced by ld-IL-2 beyond direct effects on Tregs, and relevant for further development of ld-IL-2 for therapy and prevention of T1D, and other autoimmune and inflammatory diseases.

The insulin gene in diabetes

Lack of insulin production or abnormalities affecting insulin secretion are key to the development of almost all forms of diabetes, including the common type 1 (insulin‐dependent) and type 2 (non‐insulin‐dependent) diabetes and the more rare forms of maturity‐onset diabetes of the young (MODY). Because insulin has such a central role in the pathogenesis of both forms of diabetes, the insulin gene (INS) has always been considered a candidate susceptibility gene. A number of studies have shown that the allelic variation and parent‐of‐origin effects affect the transmission and expression of the insulin gene in pancreatic β‐cells and extra‐pancreatic tissues. These observations have led to the formulation of new hypotheses to explain the biological mechanisms by which functional differences in the expression of the insulin gene may contribute to diabetes susceptibility. Copyright

The Juvenile Diabetes Research Foundation Network for Pancreatic Organ Donors with Diabetes (nPOD) Program: goals, operational model and emerging findings

nPOD actively promotes a multidisciplinary and unbiased approach toward a better understanding of T1D and identify novel therapeutic targets, through its focus on the study of human samples. Unique to this effort is the coordination of collaborative efforts and real-time data sharing. Studies supported by nPOD are providing direct evidence that human T1D isa complex and heterogeneous disease, in which a multitude of pathogenic factors may be operational and may contribute to the onset of the disease. Importantly, the concept that beta cell destruction is almost completed and that the autoimmune process is almost extinguished soon after diagnosis is being challenged. nPOD investigators are exploring the hypothesis that beta cell dysfunction may also be a significant cause of hyperglycemia, at least around the time of diagnosis, and are uncovering novel molecules and pathways that are linked to the pathogenesis and etiology of human T1D. The validation of therapeutic targets is also a key component of this effort, with recent and future findings providing new strategic direction for clinical trials.