ONLINE APPENDIX
Thirty-seven patients who received islet transplant subsequent to kidney transplant were
retrospectively studied. The patients received an islet transplant according to ABO
compatibility and a triple immunosuppressive regimen (steroids, cyclosporine, and
mofetil mycophenolate) plus induction with ATG (SANGSTAT) at the time of islet
transplant.
Patients were retrospectively split into long-term functioning (Long fx=17 patients with
C-peptide> 1.0 ng/ml for more than 12 months) or short-term functioning (Short fx=20
patients who lost C-peptide secretion> 1 ng/ml within 12 months) groups. All the data
were expressed as mean  standard error. The day of metabolic tests, subcutaneous
administration of long-acting insulin was suspended the night before and replaced with
shots of short-acting insulin.
ELISA
Human proinsulin levels were assayed with an ELISA kit with no cross-reactivity with
human insulin (DAKO Diagnostics Ltd., UK). The minimum detectable dose was 6
pmol/l. Intra-assay CV was 3.5% and inter-assay CV was 5.5%. Serum C-peptide levels
(intra-assay CV 3.0%; interassay CV 3.0%) were assayed by RIA using commercial kits
(Medical System, Genoa, Italy). Human APO-1/FAS levels were assayed with an ELISA
kit (BioSource Europe S.A., Belgium). No cross-reactivity with SIL-2R, sICAM-1,
sVCAM-1, TNF- was demonstrated, and the minimum detectable level was <2 ng/ml.
Intra-assay CV was 4.9% and inter-assay CV was 8.9%. Human sFAS Ligand levels were
assayed with an ELISA kit (Bender MedSystemsDiagnostics GmbH, Austria). The
minimum detectable dose was 0.1 ng/ml. Intra-assay CV was 6.1% and inter-assay CV
was 7.0%. Human sIL-2R levels were assayed with an ELISA kit (BioSource Europe).
The minimum detectable dose was <12 pg/ml. Intra-assay CV 5.5%, inter-assay CV
6.1%. Human Activin A levels were assayed with an ELISA kit (RD System Europe LtD,
UK). The minimum detectable dose was <1.5 pg/ml. Intra-assay CV 5.0%, inter-assay
CV 9.0%. Human Amylin levels were assayed with an ELISA kit (Peninsula
Laboratories, [name city, state USA]). No cross-reactivity with insulin, proinsulin,
glucagon, or somatostatin was demonstrated, and the minimum detectable dose was 0.05
ng/ml. An intra-assay CV of 5.0% and an inter-assay CV of 14% were demonstrated.
L-Arginine test to evaluate insulin secretory reserve
The patients were given the following tests: oral glucose tolerance test (OGTT: 75 g
glucose with samples for blood glucose and plasma free insulin at 0, 30, 60, 90, 120, 180
min); L-arginine infusion (30 g, iv, as a 10% solution given over 30 min with samples for
plasma free insulin at 0, 5, 10, 20, 30, 45, 60, 90, 120 min). These tests were performed
with the patient in a recumbent position at 09.00 h after an overnight fast. The tests were
performed at 3-6 months after transplant, 1, 2, 3, and 4 years after transplantation. Not all
the patients were given all the tests at every interval: tests were performed according to
the clinical conditions of the patient or the duration of follow-up. When islet-transplanted
patients were not insulin-free, insulin was withdrawn at least 12 h before testing.
Informed consent was obtained from all patients. Calculations: the peak of insulin
secretion after i.v. glucose was calculated as the difference between the first minute
serum free insulin values and basal values for each patient. The area under the curve
(AUC) of serum insulin levels was calculated according to the trapezoidal method for the
arginine test (delta AUC: 0–60 min).
Insulin Resistance
HOMA-IS (Homeostasis model assessment of insulin sensitivity) and HOMA-IR
(Homeostasis model assessment of insulin resistance) have been used as markers of
insulin resistance and are calculated as follows:
HOMA-IS= 22.5/[Fasting insulin (U/ml) x Fasting glycemia (mmol/l)]. The normal
value is 1. Then we calculated the HOMA-IR= 1/HOMA-IS.
Autoantibody measurements
GADAs (antibodies to glutamic acid decarboxylase) and IA-2As (autoantibodies to
protein tyrosine phosphatase isoforms IA-2) were measured using a radiobinding assay
with in vitro–translated [35S]methionine-labeled GAD65 and IA-2IC (amino acids 605–
979) as previously reported1. GADAs are expressed as arbitrary units. Results for IA-2As
were expressed as an index defined as follows: (sample counts per minute – negative
standard control counts per minute)/(positive standard control counts per minute –
negative standard control counts per minute). The thresholds for positivity were
determined from the 99th percentile of control subjects and corresponded to 3 U for
GADAs and index 0.010 for IA-2As, GADA sensitivity 84, 86, and 88%; GADA
specificity 97, 97, and 92%; IA-2A sensitivity 60, 62, and 70%; and IA-2A specificity
100, 99, and 99%, respectively. The intra- and interassay coefficients of variation of
GADA for control samples designated at 10 GADA U were 9% and 17%, respectively,
and for IA-2IC were 4.8% and 9.9%, respectively.
Statistical analysis
All the data were expressed as mean  standard error. Data were tested for normal
distribution with the Kolmogorov-Smirnov test and for homogeneity of variances with
Levene’s test. Differences between parameters were evaluated using Student’s T test
when parameters were normally distributed, Mann Whitney U test when parameters were
not normally distributed, and a chi-square test for categorical variables. Two-sided paired
Student t-test (for parametric data) and Kruskal-Wallis test (for non-parametric data)
were used to compare baseline parameters with follow-up values. Correlations were
assessed with a Spearman rank correlation coefficient. A p value of less than 0.05 (by
two-tailed testing) was considered an indicator of statistical significance. Analysis of data
was done using the SPSS statistical package for Windows (SPSS Inc., Chicago,IL).
Table 1. The two groups of patients were similar in terms of pre-transplant characteristics
and number of transplanted islets.
Long fx group
Short fx group
P value
Age (years)
39.9±1.7
38.6±1.5
0.59
Duration of diabetes
27.4±3.2
24.2±2.2
0.42
Weight (kg)
56.4±1.6
61.8±2.6
0.11
Islet Equivalent (n)
585,600±59,481
542,776±63,606
0.62
Islet Equivalent/kg (n)
10,493±1,136
8,945±1,101
0.33
(years)
References
1.
Bonifacio E, Genovese S, Braghi S, et al. Islet autoantibody markers in IDDM:
risk assessment strategies yielding high sensitivity. Diabetologia. 1995;38:816-822.