A. Secchi

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function

Aims/hypothesisEfficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation.MethodsSince 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome.ResultsBy univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071).Conclusions/interpretationThese data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

Reduction of insulin resistance by combined kidney-pancreas transplantation in Type 1 (insulin-dependent) diabetic patients

SummaryTo evaluate the effect of combined kidney and pancreas transplantation on insulin action and glucose metabolism, 15 Type 1 (insulin-dependent) diabetic patients who were undergoing combined kidney-pancreas transplantation were studied before transplantation by means of the euglycaemic hyperinsulinaemic clamp technique combined with 3-3H-glucose infusion and indirect calorimetry. Nine of the original 15 patients were studied again after four months and six after 12 months, successful combined kidney-pancreas transplantation with the same experimental protocol. Nine volunteers formed the group of normal subjects. Combined kidney-pancreas transplantation normalised hepatic glucose production and reduced peripheral insulin resistance in Type 1 diabetic uraemic patients, despite chronic immunosuppressive therapy. To further evaluate the hypothesis that residual insulin resistance was due to chronic steroid therapy, 11 additional subjects with chronic uveitis (six of whom were treated with only prednisone, and five treated only with cyclosporin) underwent the same protocol demonstrating a normal hepatic glucose production. The insulin-stimulated peripheral glucose uptake was reduced in the prednisone-treated group, but normal in cyclosporin-treated subjects. Four additional diabetic patients with a kidney transplant were also studied. They showed a peripheral insulin sensitivity intermediate between diabetic uraemic patients and patients after combined transplant. We conclude that short-term (one year) combined kidney-pancreas transplantation improves glucose metabolism by restoring normal rates of hepatic glucose production and reducing peripheral insulin resistance; chronic steroid therapy is the major determinant of residual reduced insulin action. Both kidney and pancreas substitution play a role in reducing peripheral insulin resistance.

Islet transplantation in IDDM patients.

Summary This single-centre study investigated parameters that positively correlated with the success rate after islet allotransplantation in insulin-dependent diabetic (IDDM) patients. Twenty-one intrahepatic, fresh islet transplantations were performed in 20 IDDM patients (one patient had two transplants), after or simultaneous with kidney transplantation. The correlation between number and purity of transplanted islets and final outcome was investigated. One patient died of a cardiac arrest several hours after islet transplantation; this patient was not included in the follow-up analysis. Three patients (15 %) experienced acute, irreversible, early failure of islet function, which was considered as a ’presumed rejection. Nine patients (45 %) achieved either complete insulin-independence (seven cases) or a reduction (> 50 %) of exogenous insulin requirement (two cases), with sustained serum C-peptide secretion (0.89 ± 0.04 nmol/l; duration: 21 ± 7 months, range 2–58 months). Liver biopsy, performed 3 years after transplantation in one successful case, showed normal islets within the hepatic parenchyma. Eight cases (40 %) did not show any metabolic effect of islet transplantation, with low serum C-peptide levels (’presumed function exhaustion). Metabolic investigations performed in successful cases showed an early phase of insulin release after arginine, mild and reversible postprandial hyperglycaemia and normal HbA1c levels. Success of islet transplantation positively correlates with the number (p < 0.05) of the transplanted islets. Islet transplantation is a safe procedure, with 45 % success rate, in terms of insulin-independence or relevant reduction of exogenous insulin requirement, although success can be transient. [Diabetologia (1997) 40: 225–231]

Amelioration of nerve conduction velocity following simultaneous kidney/pancreas transplantation is due to the glycaemic control provided by the pancreas

Summary Diabetic polyneuropathy is a common, disabling chronic complication of diabetes mellitus. Previous studies have suggested that combined pancreas-kidney transplantation can ameliorate nerve conduction. The relative contribution of the correction of hyperglycaemia and uraemia on nerve function is still a matter of debate. Nerve conduction velocity (NCV) was assessed before and after simultaneous pancreas and kidney transplantation, and before and after pancreas graft failure in five insulin-dependent diabetic (IDDM) patients affected by severe diabetic polyneuropathy. Sensory and motor NCV were recorded in five nerves and expressed as a cumulative index for each patient. Metabolic control was evaluated by fasting blood glucose and glycosylated haemoglobin levels. NCV index was below normal values before transplant: –3.8 ± 0.7 (normal value: 0.89), improved 1 and 2 years after transplant: –3.1 ± 1.3 and –2.6 ± 0.9 (p = 0.0019), stabilised until pancreas failure and deteriorated to pre-transplant values 2 years after pancreas graft failure: –3.6 ± 1.0 (p = 0.034). Fasting blood glucose levels worsened after pancreas graft failure. HbA1 c levels, in the normal range during functioning pancreas graft (6.6 ± 0.6 %), deteriorated after its failure (8.0 ± 0.6 %, p = 0.04). Kidney function was preserved. These data support a positive effect of pancreas transplantation per se on NCV in IDDM subjects with diabetic polyneuropathy, thus demonstrating that metabolic control provided by a self-regulated source of insulin not only halts but also ameliorates nerve function, even if polyneuropathy is advanced. [Diabetologia (1997) 40: 1110–1112]

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 melititus. 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. Patient 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. Patients 5 and 6 received a simultaneous kidney and islet graft and 6 months after transplantation their post-prandial C-peptide secretion peaks were 2.5 and 1.9 ng/ml respectively. Their daily insulin requirement was not significantly modified. In conclusion, these results show that an adequate number of human islets injected intraportally in type 1 diabetic patients can replace the pancreatic endocrine function and can lead to insulin independence.

Secretory defects induced by immunosuppressive agents on human pancreatic β-cells

Abstract. Despite the considerable interest for islet and pancreas transplantation, remarkably little is known about the direct effects of immunosuppressive drugs on human β-cell function. We measured different insulin secretory parameters and insulin gene expression of human islets cultured for 5 days in the presence of mycophenolate mofetil (MMF), cyclosporin A (CsA), tacrolimus (FK506) or a mixture of 3 cytokines. Basal insulin release after exposure to cytokines and FK506 was significantly higher than in control islets. Responsiveness to an acute glucose stimulus did not differ significantly between control and treated islets. However, absolute incremental insulin responses (Δ-AUCs) of islets exposed to cytokines or FK506 were significantly higher compared to islets exposed to CsA or MMF, mainly because of the higher basal release. Indeed, maximal over basal release (stimulation index, SI) tended to be lower in islets exposed to FK506 than in control islets. Insulin gene expression was significantly reduced only in islets exposed to CsA. FK506 was, among those tested, the immunosuppressive drug that most profoundly altered the normal insulin secretory pattern of human β-cells, whereas CsA was the only inhibiting insulin gene expression. Although the abnormalities induced by the immunosoppressive drugs utilized in this study were modest, these in vitro data are consistent with the reported in vivo diabetogenicity of CsA and FK506 and point to MMF as the ideal immunosuppressive agent from a pancreatic β-cell point of view.

Succesful transplantation of human islets in recipients bearing a kidney graft

Abstract.Aims/hypothesis: Islet transplantation is a minimally invasive approach to curing Type I (insulin-dependent) diabetes mellitus. Success has recently been reported in patients receiving solitary islet transplants but the outcome in patients receiving islets together with, or after, kidney transplants has been limited and unpredictable. Methods: Here we report successful islet transplantation in a cohort of 15 patients with Type I diabetes who were followed for at least 1 year after islet transplantation, after having already received kidney allografts because of end-stage nephropathy. Results: C-peptide after transplantation was higher than 0.17 nmol/l in all 15 recipients, reflecting the absence of primary non-function. Insulin requirement was reduced by over 50 % in all but one patient, and insulin independence was achieved in 10 (66 %) recipients, five of whom now have stable, prolonged insulin independence, well controlled fasting glycaemia, a substantial first-phase and normal second-phase response to glucose, normal insulin sensitivity (HOMA analyses) and HbA1 c of under 6.2 % (33, 26, 18, 13 and 12 months after transplantation respectively). Of importance for patient management, an assessment of fasting blood glucose and proinsulin values following overnight withdrawal of insulin administration one month after transplantation was a potent predictor of insulin independence, and could be used to decide patients who should have further islet preparations. Conclusion/interpretation: These findings support the use of islet transplantation as a cure for Type I diabetes in patients with severe complications. [Diabetologia (2002) 45: 77–84]

Persistence of counter-regulatory abnormalities in insulin-dependent diabetes mellitus after pancreas transplantation.

Abstract Conventional insulin therapy does not correct the counter‐regulatory abnormalities of insulin‐dependent diabetes mellitus. Pancreas transplantation is an alternative therapy that restores the endogenous insulin secretion in diabetes. In this study, the effects of segmental pancreas transplantation on counter‐regulation to mild hypoglycaemia were evaluated. Glucose kinetics and the counter‐regulatory hormonal responses were assessed in eight insulin‐dependent diabetics with end‐stage renal failure who had received pancreas and kidney transplantation 1 year previously, seven diabetic uraemic subjects (candidates for combined transplantation), five patients with chronic uveitis on immunosuppressive therapy comparable to pancreas recipients and 10 normal subjects. Insulin (0·3 mU kg‐1 min‐1) was infused for 2h to induce mild hypoglycaemia (plasma glucose 3·2–3·5‐mmol l‐1) and exogenous glucose was infused as required to prevent any glucose decrease below 3·1 mmol l‐1. After transplantation, two of eight recipients had hypoglycaemic episodes reported in their medical records. During the study, hepatic glucose production was rapidly suppressed in the controls and in the patients on immunsuppression (–80 ± 7 and –54 ± 7%, P < 0·001 vs. basal), and rebounded to the baseline values within 1 h (–3 ± 1 and –6 ± 2%, P= NS vs. basal). The transplant recipients had similar suppression in the first hour (–88 ± 8%, P < 0·001 vs. basal), but the suppression persisted in the second hour (–69 ± 11%, P < 0·001 vs. basal) indicating a lack of glucose counter‐regulatory response. The uraemic‐diabetics had reduced suppression of hepatic glucose production (–45 ± 14%, P < 0·001 vs. basal) with respect to the recipients (P < 0·001), but had the same lack of response in the second hour (suppression: –39 ± 12%, P < 0·001 vs. basal). In addition, the response of glucagon to hypoglycaemia was blunted in both the recipients and in the diabetic subjects. In conclusion, the alterations in glucose counter‐regulation of insulin‐dependent diabetes persists after segmental pancreas transplantation. Specifically, the increased sensitivity of hepatic glucose production to the action of insulin renders this defect more evident after transplantation.

Rapamycin Induces a Caspase‐Independent Cell Death in Human Monocytes

The immunosuppressive activity of rapamycin (RAPA) and its efficacy as an anti‐rejection agent in organ transplantation have been ascribed principally to its anti‐proliferative effects on T cells, while the activity on monocytes is partially unknown. In vitro, RAPA reduced monocyte survival by inducing a caspase‐independent cell death. RAPA‐induced monocyte cell death (RAPA‐CD) was impeded by activation of granulocyte macrophage‐colony stimulating factor family receptors or toll‐like receptor 4, and by exposure to inflammatory cytokines. In vivo, in patients who received RAPA monotherapy as part of pre‐conditioning for islet transplantation, RAPA affected survival of myeloid lineage cells. In the peripheral blood, CD33+ and CD14+ cells decreased, whereas lymphocytes appeared unaffected. In the bone marrow, myeloid precursors such as CD15+ and CD15+/CD16+ were selectively and significantly decreased, but no major cytotoxic effects were observed. The RAPA‐CD suggests a dependence of monocytes on mammalian target of RAPA pathways for nutrient usage, and this feature implies that RAPA could be selectively useful as a treatment to reduce monocytes or myeloid cells in conditions where these cells negatively affect patient, suggesting a potential anti‐inflammatory action of this drug.

Insulin secretory patterns and blood glucose homeostasis after islet allotransplantation in IDDM patients: comparison with segmental- or whole-pancreas transplanted patients through a long term longitudinal study.

IDDM patients undergoing islet, segmental pancreas or whole pancreas allotransplantation were studied at regular intervals after surgery (3–6 months, 1, 2, 3 and 4 years) to evaluate glycometabolic control (24 h metabolic profile, OGTT) and serum free insulin response to insulinogenic stimuli (arginine, IVGTT). Patients received the same immunosuppressive therapy, based on cyclosporin, steroids and azathioprine. Islet transplanted patients showed: 1) an early peak of insulin secretion after arginine, that was maintained up to 4 years; 2) an early, but low peak of insulin secretion after IVGTT, which was lost at 3 years, despite evidence that islets were still functioning (insulin independence with normal HbA1c levels); 3) a diabetic-like response to OGTT at 3 months, which improved at 2 years (IGT response); 4) fasting euglycemia with mild and reversible post-prandial hyperglycemia during the 24 h metabolic profile, which was maintained for up to 2 years. Insulin secretory patterns of islet transplanted patients were similar to segmental pancreas transplanted patients, and lower than whole pancreas transplanted patients. The reduced beta cell mass transplanted and the functional denervation of the transplanted islets seem to be the major determinants of this behaviour.