Defective glucagon secretion during hypoglycemia after intrahepatic but not nonhepatic islet autotransplantation.

Defective glucagon secretion during hypoglycemia after islet transplantation has been reported in animals and humans with type 1 diabetes. To ascertain whether this is true of islets from nondiabetic humans, subjects with autoislet transplantation in the intrahepatic site only (TP/IAT-H) or in intrahepatic plus nonhepatic (TP/IAT-H+NH) sites were studied. Glucagon responses were examined during stepped hypoglycemic clamps. Glucagon and symptom responses during hypoglycemia were virtually absent in subjects who received islets in the hepatic site only (glucagon increment over baseline = 1 ± 6, pg/mL, mean ± SE, n = 9, p = ns; symptom score = 1 ± 1, p = ns). When islets were transplanted in both intrahepatic + nonhepatic sites, glucagon and symptom responses were not significantly different than Control Subjects (TP/IAT-H + NH: glucagon increment = 54 ± 14, n = 5; symptom score = 7 ± 3; control glucagon increment = 67 ± 15, n = 5; symptom score = 8 ± 1). In contrast, glucagon responses to intravenous arginine were present in TP/IAT-H recipients (TP/IAT: glucagon response = 37 ± 8, n = 7). Transplantation of a portion of the islets into a nonhepatic site should be seriously considered in TP/IAT to avoid posttransplant abnormalities in glucagon and symptom responses to hypoglycemia.

Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010.

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999-2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p<0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007-2010 when compared to 1999-2002 (445.4±156.8 vs. 421.3±155.4×0(3) IEQ; p<0.05). Islet purity and total number of ß cells significantly improved over the study period (p<0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999-2010, and these parallel improvements in clinical outcomes over the same period.

Total pancreatectomy and islet autotransplantation in children for chronic pancreatitis: indication, surgical techniques, postoperative management, and long-term outcomes.

OBJECTIVE: Describe the surgical technique, complications, and long-term outcomes of total pancreatectomy and islet autotransplantation (TP-IAT) in a large series of pediatric patients. BACKGROUND: Surgical management of childhood pancreatitis is not clear; partial resection or drainage procedures often provide transient pain relief, but long-term recurrence is common due to the diffuse involvement of the pancreas. Total pancreatectomy (TP) removes the source of the pain, whereas islet autotransplantation (IAT) potentially can prevent or minimize TP-related diabetes. METHODS: Retrospective review of 75 children undergoing TP-IAT for chronic pancreatitis who had failed medical, endoscopic, or surgical treatment between 1989 and 2012. RESULTS: Pancreatitis pain and the severity of pain statistically improved in 90% of patients after TP-IAT (P < 0.001). The relief from narcotics was sustained. Of the 75 patients undergoing TP-IAT, 31 (41.3%) achieved insulin independence. Younger age (P = 0.032), lack of prior Puestow procedure (P = 0.018), lower body surface area (P = 0.048), higher islet equivalents (IEQ) per kilogram body weight (P = 0.001), and total IEQ (100,000) (P = 0.004) were associated with insulin independence. By multivariate analysis, 3 factors were associated with insulin independence after TP-IAT: (1) male sex, (2) lower body surface area, and (3) higher total IEQ per kilogram body weight. Total IEQ (100,000) was the single factor most strongly associated with insulin independence (odds ratio = 2.62; P < 0.001). CONCLUSIONS: Total pancreatectomy and islet autotransplantation provides sustained pain relief and improved quality of life. The ß-cell function is dependent on islet yield. Total pancreatectomy and islet autotransplantation is an effective therapy for children with painful pancreatitis that failed medical and/or endoscopic management.

Proposed thresholds for pancreatic tissue volume for safe intraportal islet autotransplantation after total pancreatectomy.

The simple question of how much tissue volume (TV) is really safe to infuse in total pancreatectomy-islet autotransplantation (TP-IAT) for chronic pancreatitis (CP) precipitated this analysis. We examined a large cohort of CP patients (n = 233) to determine major risk factors for elevated portal pressure (PP) during islet infusion, using bivariate and multivariate regression modeling. Rates of bleeding requiring operative intervention and portal venous thrombosis (PVT) were evaluated. The total TV per kilogram body weight infused intraportally was the best independent predictor of change in PP (ΔPP) (p < 0.0001; R(2) = 0.566). Rates of bleeding and PVT were 7.73% and 3.43%, respectively. Both TV/kg and ΔPP are associated with increased complication rates, although ΔPP appears to be more directly relevant. Receiver operating characteristic analysis identified an increased risk of PVT above a suggested cut-point of 26 cmH2O (area under the curve = 0.759), which was also dependent on age. This ΔPP threshold was more likely to be exceeded in cases where the total TV was >0.25 cm(3)/kg. Based on this analysis, we have recommended targeting a TV of <0.25 cm(3)/kg during islet manufacturing and to halt intraportal infusion, at least temporarily, if the ΔPP exceeds 25 cmH2O. These models can be used to guide islet manufacturing and clinical decision making to minimize risks in TP-IAT recipients.

Metabolic assessment prior to total pancreatectomy and islet autotransplant: utility, limitations and potential.

Islet autotransplant (IAT) may ameliorate postsurgical diabetes following total pancreatectomy (TP), but outcomes are dependent upon islet mass, which is unknown prior to pancreatectomy. We evaluated whether preoperative metabolic testing could predict islet isolation outcomes and thus improve assessment of TPIAT candidates. We examined the relationship between measures from frequent sample IV glucose tolerance tests (FSIVGTT) and mixed meal tolerance tests (MMTT) and islet mass in 60 adult patients, with multivariate logistic regression modeling to identify predictors of islet mass ≥2500 IEQ/kg. The acute C-peptide response to glucose (ACRglu) and disposition index from FSIVGTT correlated modestly with the islet equivalents per kilogram body weight (IEQ/kg). Fasting and MMTT glucose levels and HbA1c correlated inversely with IEQ/kg (r values -0.33 to -0.40, p ≤ 0.05). In multivariate logistic regression modeling, normal fasting glucose (<100 mg/dL) and stimulated C-peptide on MMTT ≥4 ng/mL were associated with greater odds of receiving an islet mass ≥2500 IEQ/kg (OR 0.93 for fasting glucose, CI 0.87-1.0; OR 7.9 for C-peptide, CI 1.75-35.6). In conclusion, parameters obtained from FSIVGTT correlate modestly with islet isolation outcomes. Stimulated C-peptide ≥4 ng/mL on MMTT conveyed eight times the odds of receiving ≥2500 IEQ/kg, a threshold associated with reasonable metabolic control postoperatively.

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets.

Potent induction immunotherapy promotes long-term insulin independence after islet transplantation in type 1 diabetes.

The seemingly inexorable decline in insulin independence after islet transplant alone (ITA) has raised concern about its clinical utility. We hypothesized that induction immunosuppression therapy determines durability of insulin independence. We analyzed the proportion of insulin-independent patients following final islet infusion in four groups of ITA recipients according to induction immunotherapy: University of Minnesota recipients given FcR nonbinding anti-CD3 antibody alone or T cell depleting antibodies (TCDAb) and TNF-α inhibition (TNF-α-i) (group 1; n = 29); recipients reported to the Collaborative Islet Transplant Registry (CITR) given TCDAb+TNF-α-i (group 2; n = 20); CITR recipients given TCDAb without TNF-α-i (group 3; n = 43); and CITR recipients given IL-2 receptor antibodies (IL-2RAb) alone (group 4; n = 177). Results were compared with outcomes in pancreas transplant alone (PTA) recipients reported to the Scientific Registry of Transplant Recipients (group 5; n = 677). The 5-year insulin independence rates in group 1 (50%) and group 2 (50%) were comparable to outcomes in PTA (group 5: 52%; p>>0.05) but significantly higher than in group 3 (0%; p = 0.001) and group 4 (20%; p = 0.02). Induction immunosuppression was significantly associated with 5-year insulin independence (p = 0.03), regardless of maintenance immunosuppression or other factors. These findings support potential for long-term insulin independence after ITA using potent induction therapy, with anti-CD3 Ab or TCDAb+TNF-α-i.

No islets left behind: islet autotransplantation for surgery-induced diabetes.

For patients with severe chronic pancreatitis refractory to medical interventions, total pancreatectomy can be considered to relieve the root cause of pain. The goal of a simultaneous islet autotransplant is to prevent or minimize the otherwise inevitable surgical diabetes. Islet autotransplant can successfully preserve some endogenous islet function in the majority of recipients, which mediates protection against brittle diabetes. Most maintain reasonably good glycemic control, while 30 %-40 % successfully discontinue insulin therapy. With islet autotransplants reaching a wider clinical audience, refinements in islet isolation techniques and strategies to protect islet grafts post-transplant may further improve the success of this procedure.

Quality of life improves for pediatric patients after total pancreatectomy and islet autotransplant for chronic pancreatitis.

BACKGROUND & AIMS: Total pancreatectomy (TP) and islet autotransplant (IAT) have been used to treat patients with painful chronic pancreatitis. Initial studies indicated that most patients experienced significant pain relief, but there were few validated measures of quality of life. We investigated whether health-related quality of life improved among pediatric patients undergoing TP/IAT. METHODS: Nineteen consecutive children (aged 5-18 years) undergoing TP/IAT from December 2006 to December 2009 at the University of Minnesota completed the Medical Outcomes Study 36-item Short Form (SF-36) health questionnaire before and after surgery. Insulin requirements were recorded. RESULTS: Before TP/IAT, patients had below average health-related quality of life, based on data from the Medical Outcomes Study SF-36; they had a mean physical component summary (PCS) score of 30 and mental component summary (MCS) score of 34 (2 and 1.5 standard deviations, respectively, below the mean for the US population). By 1 year after surgery, PCS and MCS scores improved to 50 and 46, respectively (global effect, PCS P < .001, MCS P = .06). Mean scores improved for all 8 component subscales. More than 60% of IAT recipients were insulin independent or required minimal insulin. Patients with prior surgical drainage procedures (Puestow) had lower yields of islets (P = .01) and greater incidence of insulin dependence (P = .04). CONCLUSIONS: Quality of life (physical and emotional components) significantly improve after TP/IAT in subsets of pediatric patients with severe chronic pancreatitis. Minimal or no insulin was required for most patients, although islet yield was reduced in patients with previous surgical drainage operations.

Predicting islet yield in pediatric patients undergoing pancreatectomy and autoislet transplantation for chronic pancreatitis.

BACKGROUND/OBJECTIVE: Chronic pancreatitis (CP) in children is associated with significant morbidity and can lead to narcotic dependence. Total pancreatectomy (TP) may be indicated in refractory CP to relieve pain; simultaneous islet autotransplant (IAT) may prevent postsurgical diabetes. About half of pediatric patients are insulin independent 1 yr after IAT. Insulin independence correlates best with the number of islets available for transplantation (islet yield). Currently there is no known method to predict islet yield in a given patient. We assessed the ability of preoperative metabolic tests to predict islet yields in 10 children undergoing TP/IAT. DESIGN/METHODS: Hemoglobin A1c (HbA(1c)) and mixed meal tolerance tests (MMTT) were obtained prior to surgery in 10 patients age

Human islet isolation for autologous transplantation: comparison of yield and function using SERVA/Nordmark versus Roche enzymes.

Islet autotransplantation (IAT) is used to preserve as much insulin-secretory capacity as possible in patients undergoing total pancreatectomy for painful chronic pancreatitis. The enzyme used to dissociate the pancreas is a critical determinant of islet yield, which is correlated with posttransplant function. Here, we present our experience with IAT procedures to compare islet product data using the new enzyme SERVA/Nordmark (SN group; n = 46) with the standard enzyme Liberase-HI (LH group; n = 40). Total islet yields (mean +/- standard deviation; 216,417 +/- 79,278 islet equivalent [IEQ] in the LH group; 227,958 +/- 58,544 IEQ in the SN group; p = 0.67) were similar. However, the percentage of embedded islets is higher in the SN group compared to the LH group. Significant differences were found in pancreas digestion time, dilution time, and digested pancreas weight between the two groups. Multivariate linear regression analysis showed the two groups differed in portal venous pressure changes. The incidence of graft function and insulin independence was not different between the two groups. The SN and LH enzymes are associated with similar outcomes for IAT. Further optimization of the collagenase/neutral protease ratio is necessary to reduce the number of embedded islets obtained when using the SN enzyme.

Prolonged insulin independence after islet allotransplants in recipients with type 1 diabetes.

We sought to determine the long-term outcomes in type 1 diabetic recipients of intraportal alloislet transplants on a modified immunosuppressive protocol. Six recipients with hypoglycemia unawareness received one to two islet infusions. Induction therapy was with antithymocyte globulin (ATG) plus etanercept for tumor necrosis factor-alpha blockade. Recipients received cyclosporine and everolimus for maintenance immunosuppression for the first year posttransplant, with mycophenolic acid or mycophenolate mofetil subsequently substituted for everolimus. Recipients have been followed for 1173 +/- 270 days since their last infusion for islet graft function (insulin independence, hemoglobin A(1c) levels and C-peptide production) and for adverse events associated with the study protocol. Of the six recipients, five were insulin-independent at 1 year, and four continue to be insulin-independent at a mean of 3.4 +/- 0.4 years posttransplant. None of the six recipients experienced recurrence of severe hypoglycemia. Measured glomerular filtration rate decreased from 110.5 +/- 21.2 mL/min/1.73 m(2) pretransplant to 82.6 +/-19.1 mL/min/1.73 m(2) at 1 year posttransplant. In conclusion, islet transplants restored insulin independence for a mean of >3 years in four of six recipients treated with ATG and etanercept induction therapy and with cyclosporine and, initially, everolimus for maintenance. Our results suggest this immunosuppressive protocol may allow long-term graft survival.

Commercially available gas-permeable cell culture bags may not prevent anoxia in cultured or shipped islets.

Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized that oxygen permeability of bags may be insufficient for islet oxygenation. We measured oxygen transmission rates through the membrane walls of three different types of commercially available bags and through SRM currently used for islet shipment. We found that the bag membranes have oxygen transmission rates per unit area about 100-fold lower than SRM. We solved the oxygen diffusion-reaction equation for 150-microm diameter islets seeded at 3000 islet equivalents per cm2, a density adequate to culture and ship an entire human or porcine islet preparation in a single gas-permeable device, predicting that about 40% of the islet volume would be anoxic at 22 degrees C and about 70% would be anoxic at 37 degrees C. Islets of larger size or islets accumulated during shipment would be even more anoxic. The model predicted no anoxia in islets similarly seeded in devices with SRM bottoms. We concluded that commercially available bags may not prevent anoxia during islet culture or shipment; devices with SRM bottoms are more suitable alternatives.

Sirolimus is associated with reduced islet engraftment and impaired beta-cell function.

Successful islet transplantation depends on the infusion of sufficiently large quantities of islets, but only a fraction of transplanted islets can survive and become engrafted, and yet the underlying mechanism remains unclear. In this study, we examined the effect of sirolimus, a key component of the immunosuppressive regimen in clinical islet transplantation, on islet engraftment and function. To distinguish the effect of sirolimus on immune rejection from its effect on islet engraftment, we used a syngeneic model. Diabetic mice were transplanted with 250 islets under the renal capsule, followed by treatment with sirolimus or vehicle for 14 days. Thirty days posttransplantation, islet grafts were retrieved for the determination of insulin content and vascular density. Compared with mock-treated controls, diabetic recipient mice receiving sirolimus exhibited impaired blood glucose profiles and reduced glucose-stimulated insulin secretion, correlating with reduced intragraft insulin content and decreased vascular density. Islets exposed to sirolimus for 24 h in culture displayed significantly diminished glucose-stimulated insulin release, coinciding with decreased pancreas duodenum homeobox-1 and GLUT2 expression in cultured islets. Furthermore, sirolimus-treated diabetic recipient mice, as opposed to mock-treated controls, were associated with dyslipidemia. These data suggest that sirolimus, administered in the early posttransplantation phase, is a confounding factor for reduced islet engraftment and impaired beta-cell function in transplants.

Reduction of early graft loss after intraportal porcine islet transplantation in monkeys.

BACKGROUND: Pig islets constitute a possible resolution to the shortage of human islets for transplantation. After intraportal infusion of porcine islets in primates, many islets are lost through what has been termed the instant blood-mediated inflammatory reaction (IBMIR). We report on our experience with IBMIR. METHODS: Ten monkeys underwent intraportal porcine islet transplantation. Immunosuppressive therapy was with conventional agents (n=3) or based on costimulation blockade (n=7). Treatment specific for IBMIR was administered in eight monkeys; two additional monkeys received no such therapy (group 1). Cobra venom factor completely inhibited complement activity in four (group 2) and dextran sulfate provided anticoagulation in four (group 3). Islet graft function was monitored by following blood glucose, insulin requirement, and porcine C-peptide values. RESULTS: In monkeys that received neither cobra venom factor nor dextran sulfate (group 1), there was rapid destruction of islets indicated by severe hypoglycemia and the need for dextrose infusion; C-peptide levels were initially low and further reduction occurred within the first five days. In both groups 2 and 3, there was significantly less destruction of islets and some reversal of diabetes. However, when 40,000 IEQ/kg were infused, normoglycemia was lost within five days, but when 80,000 IEQ/kg were infused in one case, normoglycemia was more persistent. We observed that even when C-peptide levels were in the normal range for healthy nondiabetic pigs, these were not sufficient to maintain normoglycemia in the monkeys. CONCLUSIONS: Although pretransplantation complement depletion or anticoagulation reduces porcine islet xenograft loss significantly, neither alone is sufficient to prevent IBMIR.

Gene therapy for type 1 diabetes: is it ready for the clinic?

This review, in addition to updating the growing list of type 1 diabetes- relevant gene therapies, offers an outline of short-term objectives that can readily be met to move, at least, adenoviral and adeno-associated viral-based protocols into the clinic, first as a means of facilitating islet allografts as well as platforms with which to introduce immunoregulatory transgenes. A wide array of genes have been tested to restore insulin production, to drive the differentiation of insulin-producing progenitors, and to confer immunosuppression in an antigen- and tissue-restricted manner.

National Institutes of Health-Sponsored Clinical Islet Transplantation Consortium Phase 3 Trial: Manufacture of a Complex Cellular Product at Eight Processing Facilities.

Eight manufacturing facilities participating in the National Institutes of Health-sponsored Clinical Islet Transplantation (CIT) Consortium jointly developed and implemented a harmonized process for the manufacture of allogeneic purified human pancreatic islet (PHPI) product evaluated in a phase 3 trial in subjects with type 1 diabetes. Manufacturing was controlled by a common master production batch record, standard operating procedures that included acceptance criteria for deceased donor organ pancreata and critical raw materials, PHPI product specifications, certificate of analysis, and test methods. The process was compliant with Current Good Manufacturing Practices and Current Good Tissue Practices. This report describes the manufacturing process for 75 PHPI clinical lots and summarizes the results, including lot release. The results demonstrate the feasibility of implementing a harmonized process at multiple facilities for the manufacture of a complex cellular product. The quality systems and regulatory and operational strategies developed by the CIT Consortium yielded product lots that met the prespecified characteristics of safety, purity, potency, and identity and were successfully transplanted into 48 subjects. No adverse events attributable to the product and no cases of primary nonfunction were observed.

A Multicenter Study: North American Islet Donor Score in Donor Pancreas Selection for Human Islet Isolation for Transplantation.

Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70-0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality.

Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1.

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.