Improved Human Islet Isolation Using Nicotinamide

We investigated the effects of nicotinamide (NA) supplementation of the processing medium during islet isolation. One hundred and two human pancreata were processed for clinical transplantation after preservation either in the University of Wisconsin (UW) or using the two-layer method (TLM). Pancreata were then divided into four groups and retrospectively analyzed. Group I: UW preservation followed by processing without NA, Group II: UW preservation and processing with NA, Group III: TLM preservation without NA, Group IV: TLM preservation with NA. We observed a significant increase in islet yield in Group II (4343+/-348 IEQ/g) [mean+/-SEM], compared to Group I (2789+/-348 IEQ/g) (p=0.005). Similarly, a significant increase in islet yield was observed when NA was used in the processing of organs preserved with TLM (Group IV: 5538+/-413 vs. Group III: 3500+/-629; p=0.02). Furthermore islet yield was higher in Group IV than in Group II (p<0.05). The percentages of preparations that qualified for transplantation were 25, 47, 45, 69% in Groups I, II, III, IV, respectively. Addition of NA to the processing medium significantly improved islet yields in both the UW and TLM preservation protocols, allowing for a higher percentage of islet preparations to qualify for clinical transplantation.     

Harmful Delayed Effects of Exogenous Isolation Enzymes on Isolated Human Islets: Relevance to Clinical Transplantation

The isolation process exposes human pancreatic islets to exogenous isolation enzymes. Exposure to these enzymes, as a result of intraductal injection in the pancreas or simple contact of islets with enzyme components, causes internalization into the islet cells of enzymes and their by-products. Human islets exposed to Liberase-HI exhibit a decreased insulin secretory ability that correlates with the time of exposure. This phenomenon is paralleled by increased expression of adhesion molecules (CD106 and CD62p) and activation of apoptotic pathways (Bax and Bcl-2) in islet cells. Increased functional impairment is also observed after islet transplantation in diabetic immunodeficient mice. Experimental exposure of islet grafts to exogenous isolation enzymes causes intense inflammation (CD11b positive cells) at the transplant site and it was associated with sickness behavior and eventually death of mouse recipients. The extent of these adverse effects likely deceives the standard qualitative protocols currently in use to assess islet quality in vitro. Reducing the secondary effects of exogenous isolation enzymes on isolated human islets may be crucial to enhance the quality of islets as tissue grafts.     

Improved Outcomes in Islet Isolation and Transplantation by the Use of a Novel Hemoglobin-based O2 Carrier

During isolation, islets are exposed to warm ischemia. In this study, intraductal administration of oxygenated polymerized, stroma-free hemoglobin-pyridoxalated (Poly SFH-P) was performed to improve O2 delivery. Rat pancreata subjected to 30-min warm ischemia were perfused intraductally with collagenase in oxygenated Poly SFH-P/RPMI or RPMI (control). PO2 was increased by Poly SFH-P (381.7 +/- 35.3 mmHg vs. 202.3 +/- 28.2, p = 0.01) and pH maintained within physiological range (7.4-7.2 vs. 7.1-6.6, p = 0.009). Islet viability (77% +/- 4.6 vs. 63% +/- 4.7, p = 0.04) was improved and apoptosis lower with Poly SFH-P (caspase-3: 34,714 +/- 2167 vs. 45,985 +/- 1382, respectively, p = 0.01). Poly SFH-P improved islet responsiveness to glucose as determined by increased intracellular Ca2+ levels and improved insulin secretion (SI 5.4 +/- 0.1 vs. 3.1 +/- 0.2, p = 0.03). Mitochondrial integrity was improved in Poly SFH-P-treated islets, which showed higher percentage change in membrane potential after glucose stimulation (14.7% +/- 1.8 vs. 9.8 +/- 1.4, respectively, p < 0.05). O2 delivery by Poly SFH-P did not increase oxidative stress (GSH 7.1 +/- 2.9 nm/mg protein for Poly SFH-P vs. 6.8 +/- 2.4 control, p = 0.9) or oxidative injury (MDA 1.8 +/- 0.9 nmol/mg protein vs. 6.2 +/- 2.4, p = 0.19). Time to reach normoglycemia in transplanted diabetic nude mice was shorter (1.8 +/- 0.4 vs. 7 +/- 2.5 days, p = 0.02), and glucose tolerance improved in the Poly SFH-P group (AUC 8106 +/- 590 vs. 10,863 +/- 946, p = 0.03). Oxygenated Poly SFH-P improves islet isolation and transplantation outcomes by preserving mitochondrial integrity.     

Has Time Come for New Goals in Human Islet Transplantation?

The enthusiasm regarding clinical islet transplantation has been dampened by the long-term results. Concerns about the associated risks of life-long immunosuppression and the striking imbalance between potential recipients and available donor pancreata warrant changes in some of the current goals.
Islet transplantation will never be a cure of type 1 diabetes in the majority of patients with no secondary complications, but is a valid option for a limited number of patients with brittle diabetes waiting for an organ or after organ transplantation. Furthermore, insulin independence should not be the main goal of islet transplantation, but avoidance of severe hypoglycemia and good glycemic control, which can be achieved with a relatively small functional beta-cell mass. Therefore, initially one islet infusion is sufficient. Retransplantation at a later time point remains an option, if glucose control deteriorates.
Efforts to improve islet transplantation should no longer focus on islet isolation and immunosuppression, but rather on the low posttransplant survival rate of islets caused by activation of the coagulation pathway and the limited oxygen delivery to the islets. Transplantation of smaller islets be it naturally small or size tailored reaggregated islets has the potential to facilitate these processes.     

Multicenter Analysis of Novel and Established Variables Associated with Successful Human Islet Isolation Outcomes

Islet transplantation is a promising therapy used to achieve glycometabolic control in a select subgroup of individuals with type I diabetes. However, features that characterize human islet isolation success prior to transplantation are not standardized and lack validation. We conducted a retrospective analysis of 806 isolation records from 14 pancreas‐processing laboratories, considering variables from relevant studies in the last 15 years. The outcome was defined as postpurification islet equivalent count, dichotomized into yields ≥315 000 or ≤220 000. Univariate analysis showed that donor cause of death and use of hormonal medications negatively influenced outcome. Conversely, pancreata from heavier donors and those containing elevated levels of surface fat positively influence outcome, as did heavier pancreata and donors with normal amylase levels. Multivariable logistic regression analysis identified the positive impact on outcome of surgically intact pancreata and donors with normal liver function, and confirmed that younger donors, increased body mass index, shorter cold ischemia times, no administration of fluid/electrolyte medications, absence of organ edema, use of University of Wisconsin preservation solution and a fatty pancreas improves outcome. In conclusion, this multicenter analysis highlights the importance of carefully reviewing all donor, pancreas and processing parameters prior to isolation and transplantation.     

Evaluation of Islet Transplantation from Non-Heart Beating Donors

We evaluated islet transplantation from non-heart beating donors (NHBDs) with our Kyoto Islet Isolation Method. All patients had positive C-peptide after transplantation. The average HbA(1C) levels of the five recipients significantly improved from 7.8 +/- 0.4% at transplant to 5.2 +/- 0.2% currently (p < 0.01). Three patients with no or a single autoantibody became insulin independent while the other two patients with double autoantibodies reduced their insulin requirement but did not become insulin independent. C-peptide in patients who became insulin-independent gradually increased after each transplantation whereas C-peptide in patients who did not become insulin-independent from 3 months after the first transplantation to the next transplantation dramatically decreased. The beta-score of the three patients who became insulin independent was the best of eight. In conclusion, our method makes it feasible to use NHBDs for islet transplant into type 1 diabetic patients efficiently.     

Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41–59, BMI 26–38, cold ischemic time < 10 h). Collagen IV, pan‐laminin, perlecan and laminin‐α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin‐α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan‐laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan‐laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.     

预防性添加胰蛋白酶抑制剂对成年猪胰岛分离的影响

目的　开发成年猪胰岛分离方法。方法　取成年猪胰体尾部,胰管内注入0.1%冷胶原酶(type Ⅺ)消化液, 在38.5 ℃水箱中消化后,放入4 ℃Hanks液中分离并用600μm的滤过网过滤。残留的组织重新悬浮在冷Hanks液中,并放入Ricordi’s chamber内振荡5 min后再次过滤。胰岛分离分为对照组(n=14)、Pefabloc(胰蛋白酶抑制剂)组(n=8)及FOY(胰蛋白酶抑制剂)组(n=5)。Pefabloc组和FOY组消化液中分别添加1.0 mmol/LPefabloc或FOY。结果　Pefabloc组及FOY组的胰岛收获量分别为(11 848±3 530) 个/g 和(14 496±3 693)个/g,明显高于对照组的(8 505±3 349) 个/g,P<0.05。消化后的消化液胰蛋白酶活性对照组为(114.7±50.0)BAEEU,明显高于胰管内注入前的(4. 0±1. 8) BAEEU 及Pefabloc 组的(5. 5±2. 7) BAEEU(P<0. 01),但Pefabloc组与胰管内注入前却差异无统计学意义。对照组胰岛收获量≥8 000 个/g组的胰蛋白酶活性明显低于收获量<8 000 个/g组的活性〔(78.3±26.7) BAEEU vs (137.5±48.4) BAEEU〕,P<0.05。对照组、Pefabloc组及FOY组纯化后的胰岛对不同浓度葡萄糖显示了良好的胰岛素分泌能力。结论　本实验采用的胰岛分离方法能够获得大量的猪胰岛细胞,而且预防性添加胰蛋白酶抑制剂后进一步稳定地提高了胰岛收获     

De Novo Donor‐Specific HLA Antibodies Are Associated With Rapid Loss of Graft Function Following Islet Transplantation in Type 1 Diabetes

Outcomes after islet transplantation continue to improve but etiology of graft failure remains unclear. De novo donor‐specific human leukocyte antigen (HLA) antibodies (DSA) posttransplant are increasingly recognized as a negative prognostic marker. Specific temporal associations between DSA and graft function remain undefined particularly in programs undertaking multiple sequential transplants. Impact of de novo DSA on graft function over 12 months following first islet transplant was determined prospectively in consecutive recipients taking tacrolimus/mycophenolate immunosuppression at a single center. Mixed‐meal tolerance test was undertaken in parallel with HLA antibody assessment pretransplant and 1–3 months posttransplant. Sixteen participants received a total of 26 islet transplants. Five (19%) grafts were associated with de novo DSA. Five (31%) recipients were affected: three post–first transplant; two post–second transplant. DSA developed within 4 weeks of all sensitizing grafts and were associated with decreased stimulated C‐peptide (median [interquartile range]) at 3 months posttransplant (DSA negative: 613(300–1090); DSA positive 106(34–235) pmol/L [p = 0.004]). De novo DSA directed against most recent islet transplant were absolutely associated with loss of graft function despite maintained immunosuppression at 12 months in the absence of a rescue nonsensitizing transplant. Alemtuzumab induction immunosuppression was associated with reduced incidence of de novo DSA formation (p = 0.03).     

Inflammatory Blockade Improves Human Pancreatic Islet Function and Viability

The pathogenesis of pancreatic beta-cell death in diabetes mellitus is still under investigation. Inflammation is likely to be one of the factors responsible for beta-cell death during disease development. In this study, we have used a novel antiinflammatory compound, Lisofylline (LSF), to investigate the role of inflammatory blockade in protecting human pancreatic islets. LSF is a small synthetic molecule that reduces inflammatory cytokine production and action, improves beta-cell mitochondrial metabolism, and regulates immune activities. The present study has demonstrated that the treatment of human islets with LSF not only allows the retention of glucose responsiveness and insulin secretion in the presence of multiple proinflammatory cytokines, but also enhances basal insulin secretion of beta cells in vitro. LSF also significantly reduces islet apoptosis, protects beta cells from proinflammatory cytokine damage, and maintains cellular viability. In a mouse transplantation model, insulin independence could be reached in diabetic recipient mice by implantation of 30% fewer islets when LSF was used in islet culture compared to the control group. These results demonstrate that LSF profoundly enhances beta-cell function, and suggest the potential of using inflammatory blockade, such as LSF, to improve beta-cell function for islet transplantation.     

Shipment of Human Islets for Transplantation

The use of regional human islet cell processing centers (ICPC) supporting distant clinical islet transplantation programs (CITP) has proven successful in recent clinical trials. Standardization of islet shipping protocols is needed to preserve cell product identity, quantity, quality and sterility, and to meet criteria for transplantation.We evaluated the use of gas-permeable bags for human islet preparation shipment from a single ICPC to two remote CITPs. Product release tests (counts, purity, viability, sterility and potency) were performed at both centers using identical protocols to determine adequacy for transplantation.Thirty-five islet preparations were shipped either immediately after isolation (n = 20) or following culture (n = 15). Islet recovery rate after shipment was higher in cultured preparations, when compared to those not cultured (91.2 +/- 4.9% vs. 72.9 +/- 4.7%, respectively; p < 0.05), though the overall recovery rate based on isolation and pre-transplant counts was comparable (72.9 +/- 4.7% vs. 70.4 +/- 3.5%, respectively; p = N.S.). All preparations met product release criteria for transplantation. Additional experiments showed that gas-permeable bags led to improved recovery and potency, when compared to 50-mL conical tubes or to non-gas-permeable bags for shipment.Collectively, our data demonstrate that the use of gas-permeable bags is efficient for clinical-grade and should be preferred also for the shipment of research-grade islet preparations.     

Islet alloautotransplantation: Allogeneic pancreas transplantation followed by transplant pancreatectomy and islet transplantation

Simultaneous pancreas–kidney (SPK) transplantation is an important treatment option for patients with type 1 diabetes (T1D) and end‐stage renal disease (ESRD). Due to complications, in up to 10% of patients, allograft pancreatectomy is necessary shortly after transplantation. Usually the donor pancreas is discarded. Here, we report on a novel procedure to rescue endocrine tissue after allograft pancreatectomy. A 39‐year‐old woman with T1D and ESRD who had undergone SPK transplantation required emergency allograft pancreatectomy due to bleeding at the vascular anastomosis. Islets were isolated from the removed pancreas allograft, and almost 480 000 islet equivalents were infused into the portal vein. The patient recovered fully. After 3 months, near‐normal mixed meal test (fasting glucose 7.0 mmol/L, 2‐hour glucose 7.5 mmol/L, maximal stimulated C‐peptide 3.25 nmol/L, without insulin use in the preceding 36 hours) was achieved. Glycated hemoglobin while taking a low dose of long‐acting insulin was 32.7 mmol/mol hemoglobin (5.3%). When a donor pancreas is lost after transplantation, rescue β cell therapy by islet alloautotransplantation enables optimal use of scarce donor pancreata to optimize glycemic control without additional HLA alloantigen exposure.     

Inhibition of p38 Pathway Suppresses Human Islet Production of Pro-Inflammatory Cytokines and Improves Islet Graft Function

Nonspecific inflammation is associated with primary graft nonfunction (PNF). Inflammatory islet damage is mediated at least partially by pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) produced by resident islet macrophages. The p38 pathway is known to be involved in cytokine production in the cells of the monocyte-macrophage lineage. Therefore, inhibition of the p38 pathway may prevent pro-inflammatory cytokine production by resident islet macrophages and possibly reduce the incidence of PNF. Our present study has demonstrated that inhibition of the p38 pathway by a chemical p38 inhibitor, SB203580, suppresses IL-1beta and TNF-alpha production in human islets exposed to lipopolysaccharide (LPS) and/or inflammatory cytokines. Although IL-1beta is predominantly produced by resident macrophages, ductal cells and islet vascular endothelial cells were found to be another cellular source of IL-1beta in isolated human islets. SB203580 also inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the treated islets. Furthermore, human islets treated with SB203580 for 1 h prior to transplantation showed significantly improved graft function. These results suggest that inhibition of the p38 pathway may become a new therapeutic strategy to improve graft survival in clinical islet transplantation.     

Islet Heparan Sulfate but Not Heparan Sulfate Proteoglycan Core Protein Is Lost During Islet Isolation and Undergoes Recovery Post‐Islet Transplantation

Islet beta cells in situ express intracellular heparan sulfate (HS), a property previously shown in vitro to be important for their survival. We report that HS levels inside islet beta cells correlate with the novel intracellular localization of the HSPG core proteins for collagen type XVIII (Col18), a conventional extracellular matrix component. Syndecan‐1 (Sdc1) and CD44 core proteins were similarly localized inside beta cells. During isolation, mouse islets selectively lose HS to 11–27% of normal levels but retain their HSPG core proteins. Intra‐islet HS failed to recover substantially during culture for 4 days and was not reconstituted in vitro using HS mimetics. In contrast, significant recovery of intra‐islet HS to ～40–50% of normal levels occurred by 5–10 days after isotransplantation. Loss of islet HS during the isolation procedure is independent of heparanase (a HS‐degrading endoglycosidase) and due, in part, to oxidative damage. Treatment with antioxidants reduced islet cell death by ～60% and increased the HS content of isolated islets by ～twofold compared to untreated islets, preserving intra‐islet HS to ～60% of the normal HS content of islets in situ. These findings suggest that the preservation of islet HS during the islet isolation process may optimize islet survival posttransplant.     

Cell Permeable Peptide of JNK Inhibitor Prevents Islet Apoptosis Immediately After Isolation and Improves Islet Graft Function

Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH2-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.     

Sequential Kidney/Islet Transplantation: Efficacy and Safety Assessment of a Steroid-Free Immunosuppression Protocol

The aim of this study was to assess the efficiency and safety of the Edmonton immunosuppression protocol in recipients of islet-after-kidney (IAK) grafts. Fifteen islet infusions were administered to 8 patients with type 1 diabetes and a functioning kidney graft. Immunosuppression was switched on the day of transplantation to a regimen associating sirolimus-tacrolimus-daclizumab. Insulin-independence was achieved in all patients for at least 3 months, with an actual rate of 71% at 1 year after transplantation (5 of 7 patients). After 24-month mean follow-up, five have ongoing insulin independence, 11–34 months after transplantation, with normal HbA1c, fructosamine and mean amplitude of glycemic excursions (MAGE) values. Results of arginine-stimulation tests improved over time, mostly after the second islet infusion. Severe adverse events included bleeding after percutaneous portal access (n = 2), severe pneumonia attributed to sirolimus toxicity (n = 1), kidney graft loss after immunosuppression discontinuation (n = 1), reversible humoral kidney rejection (n = 1) and fever of unknown origin (n = 1). These data indicate that the Edmonton approach can be successfully applied to the IAK setting. This procedure is associated with significant side effects and only patients with stable function of the kidney graft should be considered. The net harm versus benefit has not yet been established and will require further studies with larger numbers of enrolled subjects.     

Enhancing the Success of Human Islet Isolation Through Optimization and Characterization of Pancreas Dissociation Enzyme

A major obstacle to successful human islet isolation has been the variability of the enzymatic digestion phase. The aim of this study was to define optimal enzyme activity ranges normalized by the pancreas weight and to identify valid parameters for the optimal selection of successful lots of collagenase enzyme blends. Our results from 251 islet isolations showed that optimization of thermolysin dosage based on Caseinase unit/g pancreas contributed considerably to islet isolation outcome but that collagenase dosage measured by the manufacturer (Wünsch unit/g pancreas) was not a major determinant of islet isolation outcome. We also found that lot-to-lot inconsistency of enzyme performance was not explained by the activity values provided by the manufacturer, but rather by an in-house assay of class I collagenase (CI) and class II collagenase (CII); using a lot with a lower CII/CI resulted in a higher success rate. The odds of successful isolation was 8.67 times higher when a vial with CII/CI ratio <0.204 was used than when a vial with CII/CI >or=0.204 was used, suggesting that CII/CI ratio may be a strong predictor to distinguish potential lot success. This study provides a framework for improved enzymatic digestion in human islet isolation.