Morihito Takita

Improving efficacy of clinical islet transplantation with iodixanol-based islet purification, thymoglobulin induction, and blockage of IL-1β and TNF-α.

Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large-scale studies are necessary to confirm these results and clarify the mechanism of each component.

Seven consecutive successful clinical islet isolations with pancreatic ductal injection.

Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 ± 64,319 vs. 354,836 ± 89,649 IE, p < 0.01) and viability (97.3 ± 1.2% vs. 92.6 ± 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.

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 ×103 IEQ; p < 0.05). Islet purity and total number of β 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.

Evidence for Instant Blood-Mediated Inflammatory Reaction in Clinical Autologous Islet Transplantation

A nonspecific inflammatory and thrombotic reaction termed instant blood‐mediated inflammatory reaction (IBMIR) has been reported when allogenic or xenogenic islets come into contact with blood. This reaction is known to cause significant loss of transplanted islets. We hypothesized that IBMIR occurs in patients undergoing total pancreatectomy followed by autologous islet transplantation (TP‐AIT) and tested this hypothesis in 24 patients and in an in vitro model. Blood samples drawn during the peritransplant period showed a significant and rapid increase of thrombin–anti‐thrombin III complex (TAT) and C‐peptide during islet infusion, which persisted for up to 3 h, along with a decreased platelet count. A concomitant increase in levels of inflammatory proteins IL‐6, IL‐8 and interferon‐inducible protein‐10 was observed. An in vitro model composed of pure islets plus autologous blood also demonstrated significantly increased levels of TAT (p < 0.05), C‐peptide (p < 0.05), tumor necrosis factor‐alpha (p < 0.05) and MCP‐1 (p < 0.05), as well as strong tissue factor expression in islets. Islet viability decreased significantly but was rescued by the presence of low‐molecular‐weight dextran sulfate. In conclusion, AIT‐induced elevation of TAT and destruction of islets suggests that IBMIR might occur during AIT. Modulating this process may help improve islet engraftment and the insulin independence rate in TP‐AIT patients.

Iodixanol-controlled density gradient during islet purification improves recovery rate in human islet isolation.

Background. For pancreatic islet transplantation, islet purification minimizes the risks associated with islet infusion through the portal vein by reducing the amount of transplanted tissue. However, the purification step may result in decreased numbers of islets recovered from digested tissue and be traumatic to the islets. In this study, we evaluated the effectiveness of iodixanol-controlled density gradients on the islet purification step. Methods. For 14.3% of the isolations, the density was 1.085 g/cm3, 32.1% were 1.090 g/cm3, 46.4% were 1.095 g/cm3, 3.6% were 1.100 g/cm3, and 3.6% were 1.105 g/cm3, indicating that the density varies with each isolation. This has profound implications for the difficulty of islet purification. According to the density of digested tissue before purification, the density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto [IK] solutions). Results. Islet yield after purification and rate of postpurification recovery were significantly higher in the IK group than with standard continuous gradient purification by Ficoll solutions (islet yield=Ficoll group: 377,230±50,207 islet equivalents, IK group: 594,136±50,570 islet equivalents, P less than 0.01; percentage of recovery=Ficoll group: 55.6%±5.8%, IK group: 84.9%±4.2%, P less than 0.01). In vitro and in vivo assays suggest that the quality of islets was similar between the two groups. Conclusion. Our data suggest that using an iodixanol-controlled density gradient improves the islet recovery rate in human islet isolation. On the basis of these data, we now use this purification method for clinical islet transplantation.

Large human islets secrete less insulin per islet equivalent than smaller islets in vitro.

Islet yield is a critical parameter to determine clinical use of isolated islets. Because islet equivalent (IEQ) is used to evaluate islet yield, it is important to know the function per IEQ. In this study, we assessed insulin secretion per IEQ by our newly developed single islet glucose-stimulated insulin release test (SI-GSIRT). For SI-GSIRT, an individual islet was classified by its diameter from the area of a digital image and calculated IEQ. Each single islet was incubated with low glucose followed by high glucose solutions. Insulin secretions by SI-GSIRT were compared based on diameter of islets. There was a significantly strong correlation between insulin secretion stimulated by high glucose solution and low glucose solution (R2=0.90, p

Changes in metabolic profiles after the Great East Japan Earthquake: a retrospective observational study

BackgroundA magnitude 9.0 earthquake struck off eastern Japan in March 2011. Many survivors have been living in temporary houses provided by the local government since they lost their houses as a result of the great tsunami (tsunami group) or the expected high-dose radiation resulting from the nuclear accident at the Fukushima Daiichi Nuclear Power Plant (radiation group). The tsunami was more than 9 m high in Soma, Fukushima, which is located 30 km north of the Fukushima Daiichi Nuclear Power Plant and adjacent to the mandatory evacuation area. A health screening program was held for the evacuees in Soma in September 2011. The aim of this study was to compare the metabolic profiles of the evacuees before and after the disaster. We hypothesized that the evacuees would experience deteriorated metabolic status based on previous reports of natural disasters.MethodsData on 200 subjects who attended a health screening program in September or October of 2010 (pre-quake) and 2011 (post-quake) were retrospectively reviewed and included in this study. Pre-quake and post-quake results of physical examinations and laboratory tests were compared in the tsunami and radiation groups. A multivariate regression model was used to determine pre-quake predictive factors for elevation of hemoglobin A1c (HbA1c) in the tsunami group.ResultsSignificantly higher values of body weight, body mass index, waist circumference, and HbA1c and lower high-density lipoprotein cholesterol levels were found at the post-quake screening when compared with the pre-quake levels (p = 0.004, p = 0.03, p = 0.008, p < 0.001, and p = 0.03, respectively). A significantly higher proportion of subjects in the tsunami group with high HbA1c, defined as ≥5.7%, was observed after the quake (34.3%) than before the quake (14.8%) (p < 0.001). Regional factors, periodic clinic visits, and waist circumference before the quake were identified as predictive factors on multivariate analysis for the deterioration of HbA1c.ConclusionsPost-quake metabolic variables were impaired compared with pre-quake baseline levels in survivors who were living in temporary houses. A natural disaster could affect metabolic profiles, and careful follow-up for survivors should be planned.

Low-Temperature Preservation of Isolated Islets is Superior to Conventional Islet Culture Before Islet Transplantation

Background. Although culturing islets before transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients, it is well-known that isolated islets deteriorate rapidly in culture. In this study, we evaluated optimal temperature for culture/preservation of isolated human islets before transplantation. Methods. Isolated islets were cultured or preserved for 48 hr in the following culture/preservation conditions: preservation at 4°C in University of Wisconsin solution and culture at 22°C or 37°C in culture medium. Results. Islet morphology after 4°C preservation was similar to that of fresh islets, whereas islet diameter after 37°C or 22°C culture was smaller than that of fresh islets. Islet yield significantly decreased at higher temperatures (24% loss in 37°C culture and 19% loss in 22°C culture, but <5% loss in 4°C preservation). Cultured/preserved islets were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in the 4°C preservation group than in 22°C and 37°C culture groups. Conclusion. Preservation of isolated islets at 4°C improves the outcome of islet transplantation more efficiently than preservation at 22°C or 37°C. Based on these data, we have performed short-time cold storage of isolated islets instead of culturing for current clinical islet transplantation.

Inflammatory Response in Islet Transplantation

Islet cell transplantation is a promising beta cell replacement therapy for patients with brittle type 1 diabetes as well as refractory chronic pancreatitis. Despite the vast advancements made in this field, challenges still remain in achieving high frequency and long-term successful transplant outcomes. Here we review recent advances in understanding the role of inflammation in islet transplantation and development of strategies to prevent damage to islets from inflammation. The inflammatory response associated with islets has been recognized as the primary cause of early damage to islets and graft loss after transplantation. Details on cell signaling pathways in islets triggered by cytokines and harmful inflammatory events during pancreas procurement, pancreas preservation, islet isolation, and islet infusion are presented. Robust control of pre- and peritransplant islet inflammation could improve posttransplant islet survival and in turn enhance the benefits of islet cell transplantation for patients who are insulin dependent. We discuss several potent anti-inflammatory strategies that show promise for improving islet engraftment. Further understanding of molecular mechanisms involved in the inflammatory response will provide the basis for developing potent therapeutic strategies for enhancing the quality and success of islet transplantation.

Fresh islets are more effective for islet transplantation than cultured islets.

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.