Human insulin production and amelioration of diabetes in mice by electrotransfer-enhanced plasmid DNA gene transfer to the skeletal muscle.

A first-line gene therapy for type 1 diabetes should be based on a safe procedure to engineer an accessible tissue for insulin release. We evaluated the ability of the skeletal muscle to release human insulin after electrotransfer (ET)-enhanced plasmid DNA injection in mice. A furin-cleavable proinsulin cDNA under the CMV or the MFG promoter was electrotransferred to immune-incompetent mice with STZ-induced severe diabetes. At 1 week, mature human insulin was detected in the serum of 17/20 mice. After an initial peak of 68.5 +/- 34.9 microU/ml, insulin was consistently detected at significant levels up to 6 weeks after gene transfer. Importantly, untreated diabetic animals died within 3 weeks after STZ, whereas treated mice survived up to 10 weeks. Fed blood glucose (BG) was reduced in correspondence with the insulin peak. Fasting BG was near-normalized when insulin levels were 12.9 +/- 5.3 (CMV group, 2 weeks) and 7.7 +/- 2.6 microU/ml (MFG group, 4 weeks), without frank hypoglycemia. These data indicate that ET-enhanced DNA injection in muscle leads to the release of biologically active insulin, with restoration of basal insulin levels, and lowering of fasting BG with increased survival in severe diabetes. Therefore the skeletal muscle can be considered as a platform for basal insulin secretion.

Processing and release of human proinsulin-cleavage products into culture media by different engineered non-endocrine cells: a specific assessment by capillary electrophoresis.

The aim of this study was to compare the metabolic pathway to mature insulin through the intermediate forms (32-33 split, 65-66 split, des31,32 and des64,65) in human or murine cells engineered for the release of wild-type human proinsulin and in a genetically mutated one, in the search for a new approach for an insulin-dependent diabetes mellitus cure by gene therapy. Primary human fibroblasts, myoblasts and stabilized cell lines (HepG2 and NIH3T3) were transduced either with a retroviral vector coding for wild-type proinsulin or for a genetically mutated one, carrying cleavage sites sensitive to furin. The pattern of all the proinsulin cleavage products released into the cell culture supernatants was analyzed by capillary electrophoresis. All the cells transduced with the wild-type gene released intact proinsulin. HepG2 released a considerable amount of 65-66 split and des64,65, while primary myoblasts released all the intermediate forms and a limited amount of mature insulin. All the cells transduced with a furin-sensitive proinsulin gene released a higher amount of mature insulin (23-59% conversion yield) than the cells expressing wild-type proinsulin, whereas the total insulin was nearly constant. Only primary cells released all the cleavage products. Screening a wide variety of non-endocrine cells has revealed a large difference in the processing and release of immature and mature insulin forms, pointing to human hepatic cells as the most efficacious. Capillary electrophoresis provided on-line and in a single run a complete overview of the proinsulin metabolic pathway in different cells.

Reversal of diabetes in mice by implantation of human fibroblasts genetically engineered to release mature human insulin.

Autoimmune destruction of pancreatic beta cells in type I, insulin-dependent diabetes mellitus (IDDM) results in the loss of endogenous insulin secretion, which is incompletely replaced by exogenous insulin administration. The functional restoration provided by allogeneic beta-cell transplantation is limited by adverse effects of immunosuppression. To pursue an insulin replacement therapy based on autologous, engineered human non-beta cells, we generated a retroviral vector encoding a genetically modified human proinsulin, cleavable to insulin in non-beta cells, and a human nonfunctional cell surface marker. Here we report that this vector efficiently transduced primary human cells, inducing the synthesis of a modified proinsulin that was processed and released as mature insulin. This retrovirally derived insulin displayed in vitro biological activity, specifically binding to and phosphorylation of the insulin receptor, comparable to human insulin. In vivo, the transplantation of insulin-producing fibroblasts reverted hyperglycemia in a murine model of diabetes, whereas proinsulin-producing cells were ineffective. These results support the possibility of developing insulin production machinery in human non-beta cells for gene therapy of IDDM.

Human proinsulin production in primary rat hepatocytes after retroviral vector gene transfer.

The development of autologous somatic cells, engineered for the synthesis and release of human insulin under physiological stimuli, would certainly represent a major breakthrough in the therapy of insulin-dependent diabetes mellitus. We generated a retroviral vector containing the human proinsulin cDNA and the gene coding for the human nerve growth factor receptor for quantitative analysis of transduced cells. Primary rat hepatocytes were selected as target cells because of the constitutive expression of the pancreatic beta-cell glucose transporter GLUT-2 and the glycolitic enzyme glucokinase. Appropriate conditions for culture and retroviral transduction are described. The highest transduction efficiency, evaluated as percentage of LNGFr expressing cells was obtained by repeated infection cycles (40+/-10%). Human proinsulin accumulated in the culture medium of transduced rat hepatocytes (mean+/-SD): 18.1+/-7.9 (range 8.7-36.4) ng/24h/10(6) cells. Primary rat hepatocytes can be efficiently transduced by a retroviral vector and the de novo synthesis of human proinsulin can be induced. Primary cultured hepatocytes represent an useful model to test retroviral constructs engineered for the glucose-inducible expression of insulin under the control of liver-specific promoters.

Capillary electrophoresis for simultaneous quantification of human proinsulin, insulin and intermediate forms.

Capillary electrophoresis (CE) for the simultaneous and precise quantification of human insulin (hI), proinsulin (hPI) and intermediate forms (des 31, 32; split 65-66 and des 64, 65), released in culture media by engineered cells, is described. Analytical conditions for standard proteins were optimized using a bare silica capillary (20 cm X 50 microm internal diameter). Proteins were monitored at 200 nm and separated at constant voltage. Culture supernatants (12-24 mL) were purified on Sep-Pak Vac C18 cartridges, recovered in 1 mL of acetonitrile:trifluoracetic acid mixture (60:40, v:v), concentrated, ultrafiltered and injected into CE. Protein recovery was 85+/-14% (n = 5, mean+/-standard deviation) with a sensitivity limit of 0.5 nmol/L in the culture media, corresponding to 2 fmol injected in 22 nL. Using the CE method, it was possible to detect and quantify, with precision and accuracy, the release of hPI, hI and intermediate forms directly in the cell culture media, and to compare the proteic pattern released from engineered cells transduced with different hPI gene constructs.

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

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. HbA1c 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.

Cancer arising after pancreas and/or kidney transplantation in a series of 99 diabetic patients.

OBJECTIVE: Recipients of solid organ transplants have an increased risk of developing certain types of malignancies as compared with the general population. The majority of the literature has reported on neoplasms in kidney and heart transplant recipients. RESEARCH DESIGN AND METHODS: We describe 9 neoplasms occurring in 7 out of 73 IDDM patients after simultaneous pancreas and kidney transplantation. No cases were recorded among 26 IDDM recipients of kidney transplantation. RESULTS: Among the neoplasms found were 2 cases of posttransplant lymphoproliferative disorder (PTLD), malignant melanoma, basal-cell and squamous-cell carcinoma of the skin in the same patient, squamous-cell carcinoma in situ of the vulva, hepatocarcinoma, small-cell lung cancer, and ductal carcinoma of the breast. Four patients died. Among immunological risk factors, over-immunosuppression for steroid-resistant kidney rejection was administered only in the 2 cases of PTLD. CONCLUSIONS: Increased dosage of immunosuppressive agents may be necessary in some patients of prevent or treat rejection in view of their reduced survival on hemodialysis.

Simultaneous kidney and pancreas transplantation at the San Raffaele Scientific Institute: clinical experience and results.

Pancreas and kidney transplantation is performed in uremic IDDM patients to cure end-stage renal failure and diabetes. Seventy-two simultaneous kidney-pancreas transplantations were performed at our Institution between July 1985 and November 1994. All transplants were performed using heart-beating cadaver donors. The first 25 patients received 26 segmental pancreas according to Dubernard (KPS), whereas the last 46 patients received a whole, bladder-drained pancrea according to Sollinger (KPW). Mean pancreas cold and warm ischemia times were 294 +/- 14 and 44 +/- 2 minutes, respectively, in the KPS group and 660 +/- 37 and 40 +/- 8 minutes, respectively, in the KPW group. Twelve (48%) KPS patients and 19 (41%) KPW patients had postoperative pancreas surgical complications: vascular thrombosis led to graft failure in 5 KPS patients (20%) and 2 KPW patients (4%) (p = 0.01). Pancreatic fistula, hemorrhagic complications, and duodenum-bladder leakage were the surgical complications observed more frequently. Six KPS patients (24%) and 8 KPW patients (17%) underwent reintervention as a consequence of surgical complications. Fifteen KPS patients (60%) and 30 KPW patients (65%) experienced an acute kidney rejection episode, which was steroid-resistant in 14 KPW and 2 KPS patients. The actuarial survival rates for simultaneous kidney-pancreas recipients at one and 4 years were 92% and 84%, respectively, for KPS recipients, and 95% and 88%, respectively, for KPW patients. Kidney actuarial survival rates at one and 4 years were 96% and 76% respectively, for group KPS, and 93% and 89%, respectively, for KPW patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Octreotide administration in the treatment of pancreatic fistulae after pancreas transplantation.

Among the surgical complications of pancreas transplantation are pancreatic fistulae, which arise rather frequently. Suppression of exocrine secretion with polymers has succeeded in reducing the rate of this complication. Nevertheless, in some instances, pancreatic fistulas may occur. Thirty pancreas transplantations were performed in 27 diabetic patients. In 5 cases a pancreatic fistula occurred and was drained after the insertion of a catheter for the collection of secretions. A serous liquid was collected with a high concentration of amylases (61604 +/- 19562 IU/24 h). Fistula output was 280 +/- 87 ml/24 h. Patients were treated with octreotide, administered subcutaneously in a dose of 300-750 micrograms/day. In all patients a progressive reduction in fistula output was observed after a mean of 16 + 2 days. Fistula flow rate dropped to 24 +/- 10 ml/24 h--a reduction of 95% +/- 5% and drainage was subsequently stopped. Sonographic follow-up did not show recurrence of peripancreatic collections in these patients. All patients were insulin-independent up to 12-44 months after surgery.

Neurophysiological study of the effect of combined kidney and pancreas transplantation on diabetic neuropathy: a 2-year follow-up evaluation.

Previous study have reported a significant improvement of peripheral neuropathy following combined pancreas and kidney transplantation attributed to improvement of blood glucose control by some authors and to elimination of uraemia by others. To asses the specific role of uraemia and hyperglycaemia in neuropathy, 16 diabetic uraemic patients with combined pancreas and kidney transplantation were compared to 9 diabetic patients with a renal graft only. Neurophysiological studies of peripheral neuropathy included ulnar and deep peroneal nerve motor conduction velocity, median and sural nerve sensory conduction velocity were performed at baseline and 1 and 2 years after transplantation. One year after transplantation mean nerve conduction velocity significantly improved in both groups. However, changes were statistically significant in the kidney-pancreas group only. At the 2 year follow-up nerve conduction velocity had increased further in the pancreas-kidney group only. These data suggest that improvement of nerve conduction velocity following pancreas and kidney transplantation is predominantly due to the long-term euglycaemic state.

Pancreas and kidney transplantation: the San Raffaele Hospital (Milan, Italy) experience.

Results of 33 simultaneous pancreas and kidney transplantations performed at the San Raffaele Hospital, Milan, Italy are presented. In 26 cases segmental neoprene duct-injected grafts were transplanted and in seven cases, duodenopancreatic bladder-drained grafts. Five-year patient, kidney and pancreas survival were respectively, 89%, 72% and 58%. Five-year survival in patients with technically successful pancreas transplants was 73%. Thrombosis occurred in 20% of cases. Mortality was 6% and overall morbidity 76%. Surgical complications were present in 51% of cases.

Effect of pancreas transplantation on life expectancy, kidney function and quality of life in uraemic type 1 (insulin-dependent) diabetic patients.

The aim of our study was to evaluate the effects of haemodialysis, kidney transplantation and simultaneous kidney and pancreas transplantation on survival of diabetic subjects and on kidney function. 40 Type 1 (insulin-dependent) diabetic patients received a kidney transplantation: in 31 cases the kidney was transplanted simultaneously to a pancreas graft from the same donor (KP group), while in 9 cases the pancreas was not available (K group). 44 uraemic Type 1 (insulin-dependent) diabetic patients on dialysis and in waiting list for kidney transplantation, constituted the control group (HD group). Patient survival rate 1, 3 and 5 years following transplantation was better in KP group (93%, 89%, 89%, respectively) and in K group (88%, 88%, 73%, respectively) and in HD group (88%, 62%, 51%, respectively). Kidney graft survival at 1, 3 and 5 years post-transplant was better in KP group (93%, 72%, 72%, respectively) than in K group (76%, 61%, 31%, respectively). 1 year after transplantation, patients of the KP group who had lost the pancreas for technical reasons (thrombosis) were included in the K group so as to evaluate the effect of the transplanted pancreas on long-term patient and kidney survival. Patient survival rate in the KP group (17 patients) at 2 and 4 years was 100%, while at the same intervals it was 78% in the K group (13 patients). Kidney graft function rate at 2 and 4 years was 93% in the KP group (17 grafts) and 54% and 27% respectively in the K group (14 grafts).(ABSTRACT TRUNCATED AT 250 WORDS)