Autoantibody Response to Islet Transplantation in Type 1 Diabetes

Emanuele Bosi, Simona Braghi, Paola Maffi, Miriam Scirpoli, Federico Bertuzzi, Guido Pozza, Antonio Secchi, Ezio Bonifacio

Disclosures

Diabetes. 2001;50(11) 

In This Article

Research Design and Methods

Between December 1989 and October 2000, 37 islet transplantations were performed either simultaneously with or after kidney graft in 30 patients with long-term type 1 diabetes and renal failure at the San Raffaele Hospital Scientific Institute, Milan. In 33 of these cases performed in 27 patients (median age 40 years [range 32–61]; median duration of diabetes 22 years [9–47]) a pretransplantation serum sample and at least three posttransplantation serum samples (median sample number 6 [3–26]) were available for autoantibody measurements and were therefore included in this study.

In five patients, islets were transplanted simultaneously with kidney graft. The remaining 28 transplantations were performed after kidney graft alone or after a simultaneous pancreas and kidney graft in which pancreas failed mainly for thrombosis (median time after kidney transplantation, 21 months [1–126]).

In the five patients who received simultaneous islet and kidney transplants, islets were always from the same donor of the kidney, and in two of these patients, they were also from two additional donors; in the 28 islet transplants performed after kidney transplant, islets were transplanted from a single donor in 11, from two donors in 14, from three donors in 2, and from four donors in 1 case.

Donors were heart-beating cadavers selected for ABO blood compatibility and negative cross-match. HLA type was not considered as a criterion for the donor-recipient matching. Human islets were isolated and purified from pancreata of cadaver donors using an automated procedure as previously described[23], with subsequent modifications [24]. In all cases, islets were injected into the liver. In the first seven patients of this series, islets were injected into a branch of a mesenteric vein reached through a small midline laparotomy under general anesthesia; the remainder received islets injected directly into the portal vein, using a percutaneous transhepatic approach, under local anesthesia with continuous portal pressure monitoring. In 25 cases, patients received >6,000 islet equivalents (IEQ)/kg body wt.

Immunosuppression was based on a combination of cyclosporin and either azathioprine (18 cases) or mycophenolate (15 cases), with an additional course of 4–10 days of ALG or ATG globulin beginning immediately before the transplant procedure. A single shot of 500 mg of methylprednisolone was administered on the day of islet transplantation in 31 cases, and in 27 cases, the patient also received prednisone as part of the chronic immunosuppressive treatment. All transplantation protocols were approved by the institutional ethical committee, and all patients gave informed consent to the procedure.

In addition to the 33 patients who received islet and kidney allografts, another 3 patients (aged 22, 37, and 49 years; duration of diabetes, 14, 21, and 19 years, respectively) without diabetic nephropathy received solitary islet allografts; the transplant procedure was the same as in the kidney transplant cases, and the 3 patients received >6,000 IEQ/kg body wt from two donors in two cases and from one donor in the other. Immunosuppression was based on an experimental protocol without cyclosporin, including a single shot of 500 mg of methylprednisolone on the day of islet transplantation, ATG for 9 days in one subject (patient 1) or anti-IL2R antibodies (Simulect) on the day of islet transplantation in the other two patients, with an additional dose on day 4 in one of these; all three patients received mycophenolate and, in addition, vitamin D3 and metformin as adjuvants. The protocol was approved by the institutional ethical committee, and all patients gave informed consent for the procedure.

During the first 10 days after transplantation, the blood glucose level was kept at 5.5 to 8.2 mmol/l by continuous intravenous insulin administration and subsequently by intensified subcutaneous insulin therapy (3–4 injections/day). Insulin doses were progressively tapered according to blood glucose levels and discontinued when fasting and postprandial blood glucose levels were <6.6 and <8.8 mmol/l, respectively[25]. Patients were considered insulin-independent when these blood glucose levels were maintained without exogenous insulin for at least 1 week. In insulin-treated transplantation patients, the target level of glycated hemoglobin was <7.0%. An assessment of islet function measurement of fasting serum C-peptide was performed by radioimmunoassay (Diagnostic Products, Los Angeles, CA) daily during the first month, as long as patients remained hospitalized, and at least once 3, 6, and 12 months and every subsequent year after transplantation. The intra- and interassay coefficients of variation of C-peptide measurement were 3 and 5%, respectively.

GADA and IA-2A measurements were performed by radiobinding assay with in vitro translated 35S-methionine–labeled GAD65 or IA-2, as previously described[26,27]. Results were converted into arbitrary units by extrapolation from a standard curve with a local standard, designated as 100 units. The thresholds for positivity were determined from the 99th centile of control subjects and corresponded to 3 units for GADA and 1 unit for IA-2A. These GADA: and IA-2A assays obtained the and following performances at the First Combined Islet Autoantibody Workshop: GADA: 88% sensitivity, 98% specificity, 100% reproducibility; and IA-2A: 70% sensitivity, 99% specificity, and 100% reproducibility [22]. Antibodies to insulin (IAs) were measured using a competitive protein A/G insulin radiobinding microassay[28].

GAD epitope antibody reactivity was measured against GAD65, GAD67, and GAD65/67 chimeras by radiobinding assay as previously described[29]. The following GAD constructs were used to measure antibody binding: full-length GAD65 and GAD67, the GAD651–95/GAD67102–593 (amino-terminal GAD65 epitopes), the GAD671–101/GAD6596–234/GAD67244–593 (amino-central GAD65 epitopes), the GAD671–101/GAD65235–444/GAD67453–593 (carboxy-central GAD65 epitopes), and the GAD671–452/GAD65445–585 (carboxy-terminal GAD65 epitopes) chimeras. Radiobinding assays were carried out as for GADA.

For GADA and IA-2A IgG subclass (IgG1, IgG2, IgG3, and IgG4) and isotype (IgM, IgA, and IgE) antibody analysis, the protein A/G radiobinding assays were used, substituting the addition of the protein A/G Sepharose with IgG subclass or isotype-specific antibody-bound Sepharose beads, as previously described[30]. Results were expressed as SD scores calculated from the mean ± SD of results obtained after subtraction of nonspecific binding to beads coated with anti-rat IgM for control subjects. The mean + 3 SD was used as the threshold for detection.

Antibodies to HLA class I and class II antigens were measured by flow cytofluorimetry using a commercial kit (One Lambda, Canoga Park, CA).

Insulin independence was defined as a condition of normoglycemia in the absence of administration of exogenous insulin. The achievement of insulin independence and 95% CI were calculated by Kaplan-Meier analysis, and comparison between groups was performed using the log-rank test. For all statistical methods, the Statistical Package for Social Sciences (SPSS, Chicago, IL) was used.

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