Recurrence of Type 1 Diabetes After Simultaneous Pancreas-Kidney Transplantation, Despite Immunosuppression, Is Associated With Autoantibodies and Pathogenic Autoreactive CD4 T-Cells

ObjectiveTo investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients.Research design and methodsWe 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.ResultsAutoantibodies 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 approximately 1 year from hyperglycemia recurrence and revealed beta-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 beta-cell loss in mice receiving autoreactive T-cells but not control T-cells.ConclusionsWe demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating beta-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

Cellular Islet Autoimmunity Associates with Clinical Outcome of Islet Cell Transplantation

Islet cell transplantation can cure type 1 diabetes (T1D), but only a minority of recipients remains insulin-independent in the following years. We tested the hypothesis that allograft rejection and recurrent autoimmunity contribute to this progressive loss of islet allograft function.Twenty-one T1D patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (ATG) induction and tacrolimus plus mycophenolate mofetil (MMF) maintenance immunosuppression. Immunity against auto- and alloantigens was measured before and during one year after transplantation. Cellular auto- and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic T lymphocyte precursor assays, respectively. Humoral reactivity was measured by auto- and alloantibodies. Clinical outcome parameters--including time until insulin independence, insulin independence at one year, and C-peptide levels over one year--remained blinded until their correlation with immunological parameters. All patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. Multivariate analyses showed that presence of cellular autoimmunity before and after transplantation is associated with delayed insulin-independence (p = 0.001 and p = 0.01, respectively) and lower circulating C-peptide levels during the first year after transplantation (p = 0.002 and p = 0.02, respectively). Seven out of eight patients without pre-existent T-cell autoreactivity became insulin-independent, versus none of the four patients reactive to both islet autoantigens GAD and IA-2 before transplantation. Autoantibody levels and cellular alloreactivity had no significant association with outcome.In this cohort study, cellular islet-specific autoimmunity associates with clinical outcome of islet cell transplantation under ATG-tacrolimus-MMF immunosuppression. Tailored immunotherapy targeting cellular islet autoreactivity may be required. Monitoring cellular immune reactivity can be useful to identify factors influencing graft survival and to assess efficacy of immunosuppression.Clinicaltrials.gov NCT00623610

Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus

Background: Multiple laboratory tests are used to diagnose and manage patients with diabetes mellitus. The quality of the scientific evidence supporting the use of these tests varies substantially. Approach: An expert committee compiled evidence-based recommendations for the use of laboratory testing for patients with diabetes. A new system was developed to grade the overall quality of the evidence and the strength of the recommendations. Draft guidelines were posted on the Internet and presented at the 2007 Arnold O. Beckman Conference. The document was modified in response to oral and written comments, and a revised draft was posted in 2010 and again modified in response to written comments. The National Academy of Clinical Biochemistry and the Evidence-Based Laboratory Medicine Committee of the American Association for Clinical Chemistry jointly reviewed the guidelines, which were accepted after revisions by the Professional Practice Committee and subsequently approved by the Executive Committee of the American Diabetes Association. Content: In addition to long-standing criteria based on measurement of plasma glucose, diabetes can be diagnosed by demonstrating increased blood hemoglobin A$_{1c}$ (HbA$_{1c}$) concentrations. Monitoring of glycemic control is performed by self-monitoring of plasma or blood glucose with meters and by laboratory analysis of HbA$_{1c}$. The potential roles of noninvasive glucose monitoring, genetic testing, and measurement of autoantibodies, urine albumin, insulin, proinsulin, C-peptide, and other analytes are addressed. Summary: The guidelines provide specific recommendations that are based on published data or derived from expert consensus. Several analytes have minimal clinical value at present, and their measurement is not recommended

Autoantibodies to glutamic acid decarboxylase in palatal myoclonus and epilepsy

We report the presence of serum autoantibodies directed against glutamic acid decarboxylase in a patient with epilepsy and palatal myoclonus not associated with brain lesions. Glutamic acid decarboxylase antibody reactivity was dependent on the presence of carboxy-terminal amino acids, similar to that reported in patients with stiff-man syndrome. Marked reduction in the frequency of epileptic attacks and improvement in palatal myoclonus occurred when benzodiazepine was administered and phenytoin was gradually tapered. Testing for anti-glutamic acid decarboxylase antibodies may be indicated in patients with palatal myoclonus and with convulsive disorders refractory to therapy

Selection of donors significantly improves pig islet isolation yield

The purpose of the present study was to test the influence of donor age on islet isolation yield in swines. Large White pigs of 10 months (group 1, n = 10) and 2-3 years (group 2, n = 5) were used. A modification of the automated method for human islet isolation was used for pig islet separation. After ductal injection of a collagenase solution (Seromed, type C-1000) the islets were separated through a continuous digestion process. In group 1 it was possible to obtain an average of 636,100 islets (volume = 476.1 mm3, insulin content = 101.4 Units). In group 2 the average total islet number was 799,800 representing a volume of 1190.6 mm3 (Insulin content 210 Units). Total islet number was not significantly different in the two groups, however when islet volume and total insulin content were considered, older animals allowed a significantly higher yield (P less than 0.01 for volume and P less than 0.05 for insulin content). These findings indicate that it is possible to separate large number of pig islets from both young (10 months) and older (2-3 years) animals and that selection of donor pigs will improve islet isolation yield. These results may be of assistance in the definition of requirements for more effective islet isolation in swines

Fresh human islet transplantation to replace pancreatic endocrine function in type 1 diabetic patients. Report of six cases

The aim of this study was to evaluate the feasibility of islet allografts in patients with type 1 diabetes mellitus. Six patients received human islets from either one or two donors via the portal vein, after (n = 4) or simultaneously with (n = 2) a kidney graft. The patients with functioning kidney grafts (nos. 1-4) were already on triple immunosuppressive therapy (cyclosporine A, azathioprine, prednisone). Prednisone was increased to 60 mg/day for 15 days after the islet transplant in patient 1. Patients 2-4 and the patients who underwent a simultaneous kidney-islets graft (nos. 5, 6) also received antilymphocyte globulin. Intravenous insulin was given for the first 15 days to maintain blood glucose concentrations within the normal range. Patient 1 rejected the islets within 15 days of islet transplantation. In patient 2, a 25% reduction in insulin requirement was observed and 12 months after transplantation post-prandial serum C-peptide was 1.5 ng/ml. In patient 3, the insulin requirement decreased from 40 to 8 units/day with a post-prandial serum C-peptide of 4.1 ng/ml 12 months after islet transplantation. In patient 4 the post-prandial secretion of C-peptide increased to 6.4 ng/ml. Six months after the islet infusion, insulin therapy was discontinued and HbA1c, 24-h metabolic profile and oral glucose tolerance test remained within the normal range. He had remained off insulin for 5 months until recently, when foot gangrene paralleled a worsening of post-prandial glycaemic control. Twelve months after transplantation he is receiving 8 units insulin/day