Amyloid formation in human islets is enhanced by heparin and inhibited by heparinase.

Islet transplantation is a promising therapy for patients with diabetes, but its long-term success is limited by many factors, including the formation of islet amyloid deposits. Heparin is employed in clinical islet transplantation to reduce clotting but also promotes fibrillization of amyloidogenic proteins. We hypothesized that heparin treatment of islets during pre-transplant culture may enhance amyloid formation leading to beta cell loss and graft dysfunction. Heparin promoted the fibrillization of human islet amyloid polypeptide (IAPP) and enhanced its toxicity to INS-1 beta cells. Heparin increased amyloid deposition in cultured human islets, but surprisingly decreased islet cell apoptosis. Treatment of human islets with heparin prior to transplantation increased the likelihood of graft failure. Removal of islet heparan sulfate glycosaminoglycans, which localize with islet amyloid deposits in type 2 diabetes, by heparinase treatment decreased amyloid deposition and protected against islet cell death. These findings raise the possibility that pretransplant treatment of human islets with heparin could potentiate IAPP aggregation and amyloid formation and may be detrimental to subsequent graft function.

The challenge of pancreatic cancer therapy and novel treatment strategy using engineered mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have attracted significant attention in cancer research as a result of their accessibility, tumor-oriented homing capacity, and the feasibility of auto-transplantation. This review provides a comprehensive overview of current challenges in pancreatic cancer therapy, and we propose a novel strategy for using MSCs as means of delivering anticancer genes to the site of pancreas. We aim to provide a practical platform for the development of MSC-based therapy for pancreatic cancer.

Antiproliferation activity of Devil's club (Oplopanax horridus) and anticancer agents on human pancreatic cancer multicellular spheroids.

Devil's club (DC, Oplopanax horridus) is an important medicinal herb of the Pacific Northwest which has significant antiproliferation activity against a variety of human tumor cell lines in vitro. This study compared the antiproliferation activity of DC extract alone, and in combination with chemotherapeutic agents gemcitabine (GEM), cisplatin (CDDP), and paclitaxel (PTX) on human pancreatic cancer PANC-1 3D spheroids and 2D monolayer cells. 3D tumor spheroids were prepared with a rotary cell culture system. PANC-1 3D spheroids were significantly more resistant to killing by DC extract, GEM and PTX compared to 2D cells, with IC50 levels closer to that observed in vivo. DC extract significantly enhanced the antiproliferation activity of CDDP and GEM at some concentrations. The bioactive compound identified as a polyacetylene showed strong antiproliferation activity against PANC-1 2D cells and 3D spheroids with IC50 at 0.73±0.04 and 3.15±0.16µM, respectively. 3D spheroids and 2D cells differentially expressed a number of apoptosis related genes. Cell cycle analysis showed that the proportion of cells in S phase was increased and in G2/M phase reduced in 3D spheroids compared to 2D cells. DC extract can potentially be used to enhance the activity of chemotherapeutic agents against pancreatic cancer cells. Use of 3D spheroid model for screening of natural products can potentially increase the efficiency in discovering in vivo bioactive compounds.

The glucagon-like peptide-1 receptor agonist exenatide restores impaired pro-islet amyloid polypeptide processing in cultured human islets: implications in type 2 diabetes and islet transplantation.

AIMS/HYPOTHESIS: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), is associated with beta cell death in type 2 diabetes as well as in cultured and transplanted human islets. Impaired prohIAPP processing due to beta cell dysfunction is implicated in hIAPP aggregation. We examined whether the glucagon-like peptide-1 receptor (GLP-1R) agonist exenatide can restore impaired prohIAPP processing and reduce hIAPP aggregation in cultured human islets and preserve beta cell function/mass during culture conditions used in clinical islet transplantation. METHODS: Isolated human islets (n = 10 donors) were cultured with or without exenatide in normal or elevated glucose for 2 or 7 days. Beta cell apoptosis, proliferation, mass, function, cJUN N-terminal kinase (JNK) and protein kinase B (PKB) activation and amyloid formation were assessed. ProhIAPP, its intermediates and mature hIAPP were detected. RESULTS: Exenatide-treated islets had markedly lower JNK and caspase-3 activation and beta cell apoptosis, resulting in higher beta/alpha cell ratio and beta cell area than non-treated cultured islets. Exenatide improved beta cell function, manifested as higher insulin response to glucose and insulin content, compared with non-treated cultured islets. Phospho-PKB immunoreactivity was detectable in exenatide-treated but not untreated cultured islets. Islet culture caused impaired prohIAPP processing with decreased mature hIAPP and increased NH(2)-terminally unprocessed prohIAPP levels resulting in higher release of immature hIAPP. Exenatide restored prohIAPP processing and reduced hIAPP aggregation in cultured islets. CONCLUSIONS/INTERPRETATION: Exenatide treatment enhances survival and function of cultured human islets and restores impaired prohIAPP processing in normal and elevated glucose conditions thereby reducing hIAPP aggregation. GLP-1R agonists may preserve beta cells in conditions associated with islet amyloid formation.

TRAIL-engineered pancreas-derived mesenchymal stem cells: characterization and cytotoxic effects on pancreatic cancer cells.

Mesenchymal stem cells (MSCs) have attracted great interest in cancer therapy owing to their tumor-oriented homing capacity and the feasibility of autologous transplantation. Currently, pancreatic cancer patients face a very poor prognosis, primarily due to the lack of therapeutic strategies with an effective degree of specificity. Anticancer gene-engineered MSCs specifically target tumor sites and can produce anticancer agents locally and constantly. This study was performed to characterize pancreas-derived MSCs and investigate the effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-engineered MSCs on pancreatic cancer cells under different culture conditions. Pancreas-derived MSCs exhibited positive expression on CD44, CD73, CD95, CD105, negative on CD34 and differentiated into adipogenic and osteogenic cells. TRAIL expression was assessed by both enzyme-linked immunosorbent assay and western blot analysis. Different patterns of TRAIL receptor expression were observed on the pancreatic cancer cell lines, including PANC1, HP62, ASPC1, TRM6 and BXPC3. Cell viability was assessed using a real-time monitoring system. Pancreatic cancer cell death was proportionally related to conditioned media from MSC(nsTRAIL) and MSC(stTRAIL). The results suggest that MSCs exhibit intrinsic inhibition of pancreatic cancer cells and that this effect can be potentiated by TRAIL-transfection on death receptor-bearing cell types.

Deletion of Fas protects islet beta cells from cytotoxic effects of human islet amyloid polypeptide.

AIMS/HYPOTHESIS: Islet amyloid, which is mainly composed of human islet amyloid polypeptide (hIAPP), is a pathological characteristic of type 2 diabetes and also forms in cultured and transplanted islets. We used islet beta cells as well as two ex vivo models of islet amyloid formation, cultured human islets and hIAPP-expressing transgenic mouse islets with or without beta cell Fas deletion, to test whether: (1) the aggregation of endogenous hIAPP induces Fas upregulation in beta cells; and (2) deletion or blocking of Fas protects beta cells from amyloid toxicity. METHODS: INS-1, mouse or human islet cells were cultured with hIAPP alone, or with amyloid inhibitor or Fas antagonist. Non-transduced islets, and human islets or hIAPP-expressing mouse islets transduced with an adenovirus that delivers a human proIAPP-specific small interfering RNA (siRNA) (Ad-ProhIAPP-siRNA) were cultured to form amyloid. Mouse islets expressing hIAPP with or without Fas were similarly cultured. Beta cell Fas upregulation, caspase-3 activation, apoptosis and function, and islet IL-1ß levels were assessed. RESULTS: hIAPP treatment induced Fas upregulation, caspase-3 activation and apoptosis in INS-1 and islet cells. The amyloid inhibitor or Fas antagonist reduced apoptosis in hIAPP-treated beta cells. Islet cells with Fas deletion had lower hIAPP-induced beta cell apoptosis than those expressing Fas. Ad-ProhIAPP-siRNA-mediated amyloid inhibition reduced Fas upregulation and IL-1ß immunoreactivity in human and hIAPP-expressing mouse islets. Cultured hIAPP-expressing mouse islets with Fas deletion had similar amyloid levels, but lower caspase-3 activation and beta cell apoptosis, and a higher islet beta:alpha cell ratio and insulin response to glucose, compared with islets expressing Fas and hIAPP. CONCLUSIONS/INTERPRETATION: The aggregation of biosynthetic hIAPP produced in islets induces beta cell apoptosis, at least partially, via Fas upregulation and the Fas-mediated apoptotic pathway. Deletion of Fas protects islet beta cells from the cytotoxic effects of endogenously secreted (and exogenously applied) hIAPP.

Immunohistochemical characterisation of cells co-producing insulin and glucagon in the developing human pancreas.

AIMS/HYPOTHESIS: In adult human islets, insulin and glucagon production is largely restricted to individual cell populations. The production of these hormones is less segregated during development and during the differentiation of human pluripotent stem cells towards pancreatic lineages. We therefore sought to characterise the transcription factor profile of these cells that co-produce insulin and glucagon in the developing human pancreas, and thus to gain insight into their potential fate during normal pancreas development. METHODS: An immunohistochemical analysis was performed on human pancreas sections from fetal donors aged 9 to 21 weeks and from adult donors between the ages of 17 and 55 years. RESULTS: Endocrine cells were observed within the pancreas at all ages examined, with cells co-producing insulin and glucagon observed as early as 9 weeks of fetal age. The population of cells that co-produce insulin and glucagon generally decreased in prevalence with age, with negligible numbers in adult pancreas. From 9 to 16 weeks, the population of glucagon-only cells increased, while the insulin-only cells decreased in abundance. Cells that co-produced insulin and glucagon also produced the alpha cell transcription factor, aristaless related homeobox (ARX), and lacked the beta cell transcription factors pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1) and v-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA). CONCLUSIONS/INTERPRETATION: Our results indicate that cells co-producing insulin and glucagon in the developing human pancreas share a transcription factor profile that is similar to that of mature alpha cells and suggest that some maturing alpha cells briefly exhibit ectopic insulin expression. Thus cells that co-produce insulin and glucagon may represent a transient cell population, which gives rise to mature alpha cells.

Musashi expression in ß-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes.

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic ß-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates ß-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of ß-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and ß-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and ß-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory ß-cell proliferation and the loss of ß-cell gene expression seen in specific phases of the progression to type 2 diabetes.

Differences between amyloid toxicity in alpha and beta cells in human and mouse islets and the role of caspase-3.

AIMS/HYPOTHESIS: Type 2 diabetes is characterised by decreased beta cell mass and islet amyloid formation. Islet amyloid formed by aggregation of human islet amyloid polypeptide (hIAPP) is associated with beta cell apoptosis. We used human and transgenic mouse islets in culture to examine whether deletion of caspase-3 protects islets from apoptosis induced by endogenously produced and exogenously applied hIAPP and compared hIAPP toxicity in islet alpha and beta cells. METHODS: Human and wild-type or caspase-3 knockout mouse islet cells were treated with hIAPP. Rat insulinoma INS-1 cells were similarly cultured with hIAPP and the amyloid inhibitor Congo Red or caspase-3 inhibitor. Human and hIAPP-expressing caspase-3 knockout mouse islets were cultured to form amyloid fibrils and assessed for beta and alpha cell apoptosis, beta cell function and caspase-3 activation. RESULTS: hIAPP-treated INS-1 cells had increased caspase-3 activation and apoptosis, both of which were reduced by inhibitors of amyloid or caspase-3. Similarly, hIAPP-treated human and mouse islet beta cells had elevated active caspase-3- and TUNEL-positive cells, whereas mouse islet cells lacking caspase-3 had markedly lower beta cell but comparable alpha cell apoptosis. During culture, human islets that formed amyloid had higher active caspase-3- and TUNEL-positive beta cells than those without detectable amyloid. Finally, cultured hIAPP-expressing mouse islets lacking caspase-3 had markedly lower beta cell apoptosis than those expressing caspase-3, associated with an increase in islet beta cell/alpha cell ratio, insulin content and glucose response. CONCLUSIONS/INTERPRETATION: Prevention of caspase-3 activation protects islet beta cells from apoptosis induced by fibrillogenesis of endogenously secreted and exogenously applied hIAPP. Islet beta cells are more susceptible to hIAPP toxicity than alpha cells cultured under the same conditions.

Islet amyloid deposition limits the viability of human islet grafts but not porcine islet grafts.

Islet transplantation is a promising treatment for diabetes but long-term success is limited by progressive graft loss. Aggregates of the beta cell peptide islet amyloid polypeptide (IAPP) promote beta cell apoptosis and rapid amyloid formation occurs in transplanted islets. Porcine islets are an attractive alternative islet source as they demonstrate long-term graft survival. We compared the capacity of transplanted human and porcine islets to form amyloid as an explanation for differences in graft survival. Human islets were transplanted into streptozotocin-diabetic immune-deficient mice. Amyloid deposition was detectable at 4 weeks posttransplantation and was associated with islet graft failure. More extensive amyloid deposition was observed after 8 weeks. By contrast, no amyloid was detected in transplanted neonatal or adult porcine islets that had maintained normoglycemia for up to 195 days. To determine whether differences in IAPP sequence between humans and pigs could explain differences in amyloid formation and transplant viability, we sequenced porcine IAPP. Porcine IAPP differs from the human sequence at 10 positions and includes substitutions predicted to reduce its amyloidogenicity. Synthetic porcine IAPP was considerably less amyloidogenic than human IAPP as determined by transmission electron microscopy, circular dichroism, and thioflavin T binding. Viability assays indicated that porcine IAPP is significantly less toxic to INS-1 beta cells than human IAPP. Our findings demonstrate that species differences in IAPP sequence can explain the lack of amyloid formation and improved survival of transplanted porcine islets. These data highlight the potential of porcine islet transplantation as a therapeutic approach for human diabetes.

Impaired proinsulin processing is a characteristic of transplanted islets.

We sought to determine whether recipients of islet transplants have defective proinsulin processing. Individuals who had islet allo- or autotransplantation were compared to healthy nondiabetic subjects. Insulin (I), total proinsulin (TP), intact proinsulin and C-peptide (CP) were measured in samples of fasting serum by immunoassay, and the ratios of TP/TP+I and TP/CP were calculated. Islet allotransplant recipients had elevated TP levels relative to nondiabetic controls (16.8 [5.5-28.8] vs. 8.4 [4.0-21.8] pmol/L; p < 0.05) and autologous transplant recipients (7.3 [0.3-82.3] pmol/L; p < 0.05). Islet autotransplant recipients had significantly higher TP/TP+I ratios relative to nondiabetic controls (35.9 +/- 6.4 vs. 13.9 +/- 1.4%; p < 0.001). Islet allotransplant recipients, some of whom were on insulin, tended to have higher TP/TP+I ratios. The TP/CP ratio was significantly higher in both islet autotransplant (8.9 [0.6-105.2]; p < 0.05) and allotransplant recipients (2.4 [0.8-8.8]; p < 0.001) relative to nondiabetic controls (1.4 [0.5-2.6]%). Consistent with these findings, TP/TP+I and TP/CP values in islet autotransplant recipients increased significantly by 1-year posttransplant compared to preoperative levels (TP/CP: 3.8 +/- 0.6 vs. 23.3 +/- 7.9%; p < 0.05). Both allo- and autotransplant subjects who received <10,000 IE/kg had higher TP/CP ratios than those who received >10,000 IE/kg. Islet transplant recipients exhibit defects in the processing of proinsulin similar to that observed in subjects with type 2 diabetes manifest as higher levels of total proinsulin and increased TP/TP+I and TP/CP ratios.

Functional assessment of microencapsulated porcine islets with agarose polystyrene sulfonic acid in vitro and in xenotransplantation.

We evaluated the functional efficacy of microencapsulated porcine islet xenografts transplanted into nonobese diabetic (NOD) mice. Islets were isolated from the pancreata of CSK miniature swine by manual collagenase digestion and Ficoll purification. Purified porcine islets were immediately encapsulated into microbeads of agarose polystyrene sulfonic acid (Ag-PSSa). They remained morphologically intact by dithizone staining after 7 days in culture. Insulin secretion from encapsulated islets was determined in response to glucose challenge during perifusion. When encapsulated islets were exposed to 200 mg/dL glucose, within 5 minutes, insulin release became 5-fold greater than that at 80 mg/dL. However, a second phase insulin secretion appeared in response to 250 mg/dL glucose challenge. In xenotransplantation, microencapsulated porcine islets (1000 to 1800 MC islets) were transplanted into the peritoneal cavity of diabetic NOD mice (n = 4) without immunosuppression. The survival times after the onset of diabetes were observed after both MC islets transplanted NOD mice and nontransplanted NOD mice (n = 4). MC islets transplant recipients had significantly (P < .05) longer survival (47.5 +/- 18.6; mean +/- SD) than nontransplanted NOD mice (21.0 +/- 9.31), although random blood glucose levels were not normalized.

Serine-protease inhibition during islet isolation increases islet yield from human pancreases with prolonged ischemia.

BACKGROUND: Islet isolation from the pancreatic tissue matrix remains highly variable. Recent evidence suggests that intrinsic human pancreatic proteases, including trypsin, may inhibit effective collagenase enzymatic activity during islet isolation, thereby impairing the isolation success. In this study we have hypothesized that serine protease inhibition applied during pancreatic digestion, could improve yield and/or functional viability of islets isolated from human pancreases. METHODS: Twelve organ donor pancreases with 12.9+/-0.6 hr cold storage (mean+/-SEM) were perfused via their ducts with Liberase-HI enzyme in the presence (n=6) or absence (n=6) of 0.4 mM Pefabloc. All were then gently dissociated and their purified islets separated with Ficoll density gradient centrifugation. RESULTS: Donor-related factors (age, gender, cold storage time, body mass index, and pancreas weight) did not differ significantly between the two experimental groups. Pefabloc supplementation did not affect the digestion time, islets remaining trapped in exocrine tissue, or final islet purity. Islet recovery was increased in the Pefabloc-treated group (mean+/-SEM yield 323.8+/-80.8 x 10(3) islet equivalents vs. 130.8+/-13.6 x 10(3) islet equivalents, P<0.05). Cellular composition, DNA and insulin content, and insulin secretory activity of the isolated islets was similar. CONCLUSIONS: Inhibition of intrinsic protease activity within pancreases after prolonged cold storage improves isolation of viable islets.

Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.

BACKGROUND: Registry data on patients with type 1 diabetes mellitus who undergo pancreatic islet transplantation indicate that only 8 percent are free of the need for insulin therapy at one year. METHODS: Seven consecutive patients with type 1 diabetes and a history of severe hypoglycemia and metabolic instability underwent islet transplantation in conjunction with a glucocorticoid-free immunosuppressive regimen consisting of sirolimus, tacrolimus, and daclizumab. Islets were isolated by ductal perfusion with cold, purified collagenase, digested and purified in xenoprotein-free medium, and transplanted immediately by means of a percutaneous transhepatic portal embolization. RESULTS: All seven patients quickly attained sustained insulin independence after transplantation of a mean (+/-SD) islet mass of 11,547+/-1604 islet equivalents per kilogram of body weight (median follow-up, 11.9 months; range, 4.4 to 14.9). All recipients required islets from two donor pancreases, and one required a third transplant from two donors to achieve sustained insulin independence. The mean glycosylated hemoglobin values were normal after transplantation in all recipients. The mean amplitude of glycemic excursions (a measure of fluctuations in blood glucose concentrations) was significantly decreased after the attainment of insulin independence (from 198+/-32 mg per deciliter [11.1+/-1.8 mmol per liter] before transplantation to 119+/-37 mg per deciliter [6.7+/-2.1 mmol per liter] after the first transplantation and 51+/-30 mg per deciliter [2.8+/-1.7 mmol per liter] after the attainment of insulin independence; P<0.001). There were no further episodes of hypoglycemic coma. Complications were minor, and there were no significant increases in lipid concentrations during follow-up. CONCLUSIONS: Our observations in patients with type 1 diabetes indicate that islet transplantation can result in insulin independence with excellent metabolic control when glucocorticoid-free immunosuppression is combined with the infusion of an adequate islet mass.

Frontiers in transplantation of insulin-secreting tissue for diabetes mellitus.

Transplantation of insulin-secreting tissue represents a physiologic approach to reverse diabetes mellitus. Pancreas transplants yield a remarkable enhancement in quality of life and appear to modify the devastating neurovascular complications of diabetes. A more attractive approach is transplantation of insulin-secreting cells, a procedure of low invasiveness with the exciting prospect of modulating graft immunogenicity before transplantation, so as to minimize requirements for toxic immunosuppressive drugs. The Surgical-Medical Research Institute at the University of Alberta in Edmonton, and several others centres throughout the world, has demonstrated that islet cell transplants can reverse insulin dependence and induce remarkable glycemic stability for several years. However, widespread success has been denied because of insufficient donor tissue, early failures to reverse insulin dependence and the loss of graft function with time. Promising new research approaches to these problems are reviewed, including xenogeneic sources of cells, engineering islet cells with genes that induce expression of immunoprotective molecules, and neogenesis factors that may sustain populations of transplanted beta cells.

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.