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To resolve the critical donor shortage worldwide, enlarging the potential donor pool to include expanded criteria donors is necessary. Despite numerous attempts to establish new preservation solutions, no dramatic innovation has occurred since University of Wisconsin (UW) solution displaced Euro Collins' solution; UW solution remains the global gold standard. We previously developed a heavy water (D 2 O)-containing organ storage solution, Dsol, which is effective for livers subjected to extended cold storage (CS), and reported its effectiveness. Dsol is a modified UW solution; however, the substances or conditions that exhibit a synergistic or additive effect with D 2 O are unclear. Here we made UWD solution by removing hydroxyethyl starch (HES) from and adding 30%-D 2 O to UW solution, and compared the effects of these solutions. After 48 hours of CS, the livers were reperfused at 37 °C on an isolated perfused rat liver apparatus, and their perfusion kinetics, functions, and injuries were compared. In the UW group, portal vein resistance significantly increased and the oxygen consumption rate and bile production decreased; in contrast, these changes were suppressed in the UWD group. Organ expansion and liver damage progressed in both groups. These results confirmed that the removal of HES from and addition of D 2 O to the UW solution reduced CS-induced cellular function impairments and microcirculatory disorders. However, to reduce injury during reperfusion after CS, it is necessary to provide conditions that inhibit injury progression after reperfusion., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Moto Fukai reports financial support was provided by Japan Society for the Promotion of Science. Tsuyoshi Shimamura reports financial support was provided by Japan Society for the Promotion of Science. Moto Fukai reports a relationship with Japan Society for the Promotion of Science that includes: funding grants. Tsuyoshi Shimamura reports a relationship with Japan Society for the Promotion of Science that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

BackgroundA recent study by Chen et al described a therapy for diabetes that involved electroporation of primary hepatocytes with human proinsulin cDNA, p3MTChins. Intrahepatic transplantation of treated hepatocytes into streptozotocin (STZ) murine and porcine models led to euglycemia, weight maintenance, and normal insulin production. We tested the repeatability of their basic experiments and transplantation technique and expanded the study to include an autoimmune model.MethodsHepatocytes were isolated from B6 mice, electroporated with p3MTChins, and glucose-challenged or were injected into hepatic or spleen parenchyma of STZ-diabetic B6 and non-obese diabetic mice. Outcomes included survival, serum glucose levels, insulin, and c-peptide release. Untransfected primary hepatocytes and mice transplanted with these cells served as controls.Resultsp3MTChins-hepatocytes secreted insulin during glucose challenge, but glucose levels did not change with increasing glucose concentrations. Direct hepatic injection led to high mortality rates. Mice that underwent intrasplenic injection survived for >50 days (control = 4 days) and had a mild but stable improvement in hyperglycemia. C-peptide in both mouse models was detectable but eventually declined to baseline in the non-obese diabetic mice.ConclusionsHepatocytes can be transfected with p3MTChins to produce human insulin but may lack the proper glucose-sensing or complex storage and secretion capabilities that allow for a finely tuned dynamic insulin response. Treatment is subtherapeutic, and p3MTChins-hepatocyte function may not endure in an autoimmune model. Without successful preliminary findings, cell therapy involving electroporation of p3MTChins does not appear to be practical as a therapy for diabetes and may not be a strategy to pursue at this time.

BackgroundThe shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment).MethodIE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function.

The present study aims to demonstrate the protective effect of bone marrow mesenchymal stem cells (bmMSCs) on transplanted islets and its potential therapeutic role of severe acute pancreatitis (SAP) in rat model. Mesenchymal stem cells (MSCs) were isolated from 6 male SD rats, and were identified. The Islets isolated from 20 SD rats were evenly and randomly divided into co-culture group, and basic culture group (control group), in which the islets were cultured in DMEM/F12 medium, so as to compare the insulin secretion and stimulation index. Severe AP was induced in SD rats by retrograde injection of sodium taurocholate. Ninety rats were randomly and evenly assigned into 5 groups: control group (healthy rats), SAP group, tail vein injection group, intraperitoneal injection group and combined injection (tail vein + intraperitoneal) group. Rats were sacrificed on day 1, 2, and 3. The pancreatic tissues and blood were collected. The plasma levels of IL-10, IL-1β, TNF-α, IL-6 were determined using ELISA. Pathologic changes of the pancreas were observed using HE staining, and the positioning of DAPI labeled bmMSCs in vivo were detected. Insulin secretion and the stimulation index of co-culture group were significantly higher than those of basic culture group (P <.05), after 7 and 14 days of culture. Inflammation, edema, hemorrhage and necrosis in each model of pancreatitis were reduced significantly in BMMSCs injection group as compared to SAP group (P <. 05). Infused BMMSCs through combined injection indicated improved outcome than that of tail-vein injection or intraperitoneal injection alone. Co-culture of BMMSCs with transplanted islets prolongs the survival time of islets and maintains in vitro activity. In the rat model of SAP, combined injection of BMMSCs through tail vein and intraperitoneal significantly suppresses the inflammatory reaction and alleviates pancreatic injury in rat SAP model. • It is important to develop an efficient and safe method to isolate MSCs, and to repair and protect the isolated islets, so as to prolong the survival of the transplanted islets. • The results of this study showed that the combined culture of BMMSCs and transplanted islets has a protective effect on the transplanted islets. • In the rat model of SAP, combined injection of BMMSCs through tail vein and intraperitoneal significantly suppresses the inflammatory reaction and alleviates pancreatic injury in rat SAP model. [ABSTRACT FROM AUTHOR]

Background: Histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW) solutions are the two primary solid-organ preservation solutions used in the United States (>95%), but flush volumes vary markedly by region and center. This study analyzes data from a single organ procurement organization (OPO) to determine the actual clinical flush volumes used for HTK and UW for liver and pancreas grafts., Methods: All procurements at Indiana Donor Network were analyzed (2016-2020), and data were extracted from the on-site records. Variables included procuring center, solution, volumes, and vessels flushed. Brand and generic versions were considered equivalent. No clinical transplant outcomes were available., Results: Data were analyzed from 875 liver and 192 pancreas procurements by over 70 U.S. centers representing 10 of 11 UNOS regions. The large majority of liver grafts were preserved with HTK (n=810, 93%; UW n=93, 7%). All liver donors received an aortic flush (100%), while portal vein flush was 14% in-situ and 88% back table. For liver grafts, the median volume of infused solution was less for HTK when compared to UW (4225mL vs 5500mL, p=0.04). For pancreas procurement, 100% received aortic flush of the graft, with median HTK and UW volumes being equivalent (3000mL; p=0.85). Pediatric organs were flushed with markedly higher weight-based volumes., Conclusions: Flush volumes for HTK and UW are similar at one midwestern OPO, with data comprised of procurements performed by centers from across the U.S. These data demonstrate that low-volume HTK flush is commonly used, and this practice may be considered as a cost-saving measure., Competing Interests: Declaration of Competing Interest Dr. Richard S. Mangus has received travel support and honoraria from F. Kohler-Chemie (Germany) for speaking engagements regarding the use of preservation solutions. This company produces and distributes HTK. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Abstract Introduction Pancreas transplantation is the most effective and physiological method of treatment of patients with diabetes. It restores proper insulin secretion and helps to achieve a metabolic balance by eliminating the need for exogenous insulin. It can also prevent life-threatening diabetic complications. Pancreas transplantation can also reduce cardiovascular risk factors and can prevent the evolution of some of the chronic diabetic complications. Patients and Methods From November 2004 until September 2017, 193 pancreas transplantations were performed in the Clinical Department of Gastroenterological Surgery and Transplantation in Central Clinical Hospital of the Ministry of the Interior and Administration in Warsaw. All medical records of the recipients who underwent pancreas transplantation in our center were retrospectively analyzed. Results Among 193 transplantations, we performed 159 simultaneous pancreas kidney transplantations (SPK), 25 pancreas transplantations alone (PTA), and 9 pancreas after kidney transplantations (PAK). The overall patient survival was 87% at 1 year and 85% at 3 and 5 years after transplantation. At 1 year after transplantation, 72% patients had a fully functioning pancreas graft; at 3 years, 68%; and at 5 years, 63%. Kidney graft survival was 87% at 1 year and 85% at 3 and 5 years after transplantation. Conclusions Pancreas transplantation is an established treatment for long-lasting diabetes. It is a complicated procedure, which requires experience and multidisciplinary approach. It is an operation with a relatively high complication rate and should be performed in highly specialized centers. Highlights • Pancreas transplantation is a complicated procedure with a high complication rate. • Pancreas transplantation requires experience and a multidisciplinary approach and should be performed in highly specialized centers. • Great care and alertness is especially necessary within the first year after the pancreas transplantation, as the risk of graft failure or morbidity is the highest. [ABSTRACT FROM AUTHOR]

Background: The evidence supporting intensive blood glucose control to prevent surgical site infections (SSIs) among liver transplant recipients is insufficient. We aimed to assess the effects of postoperative intensive blood glucose control (IBGC) against standard blood glucose control (SBGC) on the incidence of SSIs among adult liver transplant recipients., Methods: We performed a randomized controlled trial (ClinicalTrials.gov identifier NCT03474666). The IBGC target was 80 to 130 mg/dL, and the SBGC target was below 180 mg/dL. Analyses were made on an intention-to-treat basis., Results: Of the 41 recipients enrolled onto the trial, 20 were randomly allocated to the IBGC group and 21 to the SBGC group. There were no significant differences in SSIs among recipients allocated to either group (relative risk [RR], 0.78; 95% confidence interval [CI], 0.21-2.88; P = .69). Mean (SD) blood glucose levels were significantly lower in the IBGC group in the 24-hour period after surgery (145.0 [20.7] mg/dL and 230.2 [51.6] mg/dL; P = .001). While there were fewer episodes of hypoglycemia in the IBGC group, this was not statistically significant. There were no episodes of severe hypoglycemia in either group. Hyperglycemia and severe hyperglycemia were significantly more frequent in the SBGC group (RR, 0.70; 95% CI, 0.52-0.93; P = .001 and RR, 0.07; 95% CI, 0.01-0.48; P = .001, respectively). Length of hospital stay was significantly shorter for recipients in the IBGC group (13.1 [5.5] days vs 19.3 [12.1] days; P = .04)., Conclusions: Although this small trial did not find intensive control reduced SSI, it was associated with lower blood glucose levels, fewer episodes of hyperglycemia and severe hyperglycemia, and shorter length of hospital stay., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Human islet allotransplantation for the treatment of type 1 diabetes is in phase III clinical trials in the U.S. and is the standard of care in several other countries. Current islet product release criteria include viability based on cell membrane integrity stains, glucose-stimulated insulin release, and islet equivalent (IE) dose based on counts. However, only a fraction of patients transplanted with islets that meet or exceed these release criteria become insulin independent following 1 transplant. Measurements of islet oxygen consumption rate (OCR) have been reported as highly predictive of transplant outcome in many models. Method: In this article we report on the assessment of clinical islet allograft preparations using OCR dose (or viable IE dose) and current product release assays in a series of 13 first transplant recipients. The predictive capability of each assay was examined and successful graft function was defined as 100% insulin independence within 45 days post-transplant. Results: OCR dose was most predictive of CTO. IE dose was also highly predictive, while glucoses stimulated insulin release and membrane integrity stains were not. Conclusion: OCR dose can predict CTO with high specificity and sensitivity and is a useful tool for evaluating islet preparations prior to clinical human islet allotransplantation. [ABSTRACT FROM AUTHOR]

Abstract: Engineered pseudoislets reconstituted from a suspension of pancreatic α and β cells have the potential to relieve the shortage of donor islets for transplantation in the treatment of type 1 diabetes. However, the methods to fabricate pseudoislets are not well developed. In this study, we attempted to generate pseudoislets, which show a higher potential for glucose-induced insulin secretion, by altering total cell number, adjusting the cell ratio of pancreatic α and β cells, and fabricating microchannel networks with the use of alginate hydrogel beads. To effectively aggregate α and β cells and hydrogel beads, we used a previously established rapid aggregation method. When pseudoislets were reconstituted with 8,000 cells in a 1:8 α/β-cell ratio, we observed that the glucose-induced insulin secretion was enhanced by 3.1 times compared with the pseudoislets formed with β cells only. In addition, embedding of microchannel networks increased the insulin secretion rate by 4.4 times compared with the pseudoislets without the microstructures. These findings demonstrated that active modification was effective in reconstituting higher functional pseudoislets, which may be useful for islet transplantation. [Copyright &y& Elsevier]

Abstract: Type I diabetes mellitus (TID) results from the autoimmune destruction of the insulin-producing pancreatic β-cells. Gene therapy is one strategy being actively explored to cure TID by affording non–β-cells the ability to secrete insulin in response to physiologic stimuli. In previous studies, we used a novel surgical technique to express furin-cleavable human insulin (INS-FUR) in the livers of streptozotocin (STZ)–diabetic Wistar rats and nonobese diabetic (NOD) mice with the use of the HMD lentiviral vector. Normoglycemia was observed for 500 and 150 days, respectively (experimental end points). Additionally, some endocrine transdifferentiation of the liver, with storage of insulin in granules, and expression of some β-cell transcription factors (eg, Pdx1, Neurod1, Neurog3, Nkx2-2, Pax4) and pancreatic hormones in both studies. The aim of this study was to determine if this novel approach could induce liver to pancreatic transdifferentiation to reverse diabetes in pancreatectomized Westran pigs. Nine pigs were used in the study, however only one pig maintained normal fasting blood glucose levels for the period from 10 to 44 days (experimental end point). This animal was given 2.8 × 109 transducing units/kg of the lentiviral vector expressing INS-FUR. A normal intravenous glucose tolerance test was achieved at 30 days. Reverse-transcription polymerase chain reaction analysis of the liver tissue revealed expression of several β-cell transcription factors, including the key factors, Pdx-1 and Neurod1, pancreatic hormones, glucagon, and somatostatin; however, endogenous pig insulin was not expressed. Triple immunofluorescence showed extensive insulin expression, as was previously observed in our studies with rodents. Additionally, a small amount of glucagon and somatostatin protein expression was seen. Collectively, these data indicate that pancreatic transdifferentiation of the liver tissue had occurred. Our data suggest that this regimen may ultimately be used clinically to cure TID, however more work is required to replicate the successful reversal of diabetes in increased numbers of pigs. [Copyright &y& Elsevier]

Abstract: Cyclosporine (CsA) and sirolimus (SRL) have been associated with undesirable side effects, including posttransplantation diabetes and hyperlipidemia, but the molecular mechanisms underlying these effects remain to be elucidated. Animal studies focusing on clinically relevant doses are advised. This study sought to compare the metabolic effects on isolated rat adipocytes treated with either CsA or SRL ex vivo and after long-term in vivo treatment in Wistar rats. We assessed the ex vivo effects of CsA (0.5–30 μmol/L) and SRL (1–250 μmol/L) on insulin-stimulated 14C-glucose uptake in epididymal adipocytes (n = 6–9). In parallel, rats (n = 12) were treated with either vehicle, CsA (5 mg/kg/d) or SRL (1 mg/kg/d) for either 3 or 9 weeks. At the end of the treatment, glucose tolerance test (GTT) and insulin-stimulated 14C-glucose uptake as well as biochemical parameters were analyzed. A significant reduction in the insulin-stimulated glucose uptake over basal was observed among isolated adipocytes, whether exposed ex vivo or in vivo to CsA or SRL treatment. Furthermore, the SRL group showed significantly lighter fat pads and smaller adipocytes at 3 weeks with a smaller gain in body weight throughout the study compared with either the vehicle or CsA cohorts. Glucose intolerance was observed after a GTT, at the end of the treatment with either drug. Additionally, at 9 weeks serum triglycerides were increased by CsA compared with vehicle or SRL treatment. Interestingly, although SRL-treated animals presented higher fed and fasted insulin levels compared with either group, suggesting insulin resistance, the CsA group presented lower fed and fasted insulin values, suggesting a defect in insulin secretion at 9 weeks. These results suggested that either ex vivo treatment of fat cells or in vivo treatment of rats with CsA or SRL impaired insulin-stimulated glucose uptake by adipocytes. Both drugs caused glucose intolerance, which altogether could be responsible for the development of posttransplantation diabetes. [Copyright &y& Elsevier]

Abstract: Introduction: Although Islet cell isolation and culture have been well developed, there has been little progress to prolong transplanted islet sruvival. Hepatic ischemia and insufficient neovascularization of islets are considered to be the barriers to long-term survival, Hepatocytes that survive ischemic injury have been reported to protect themeslves and regenerate using the IL-6 interleukin 6 and STAT3 pathways. Materials and Methods: The hepatocellular carcinoma (Hep-G2) cell line preconditioned for 0, 2, 4, 6, and 24 hours in a hypoxic chamber, was cocultured with rat insulin-secreting celline (RIN-5F) cells. We measured cell viabilities, insulin secretion, and p-STAT3, IL-6, and NF-κB levels. Results: Cocultured Hep-G2 and RIN-5F cells aggregated to form spheroids. Viabilities of Hep-G2 cells were no different after various ischemic preconditioning times, but insulin secretion increased in a time-dependent fashion with preconditioning. Western blotting showed p-STAT3, NF-κB, and IL-6 levels to increase with preconditioning time. Conclusion: The IL-6/STAT3 pathway of Hep-G2 cells after ischemic injury showed beneficial effects on insulin secretion of RIN-5f cells cocultured with themselves. [Copyright &y& Elsevier]

Abstract: During islet transplantation into the portal vein of the liver, the islet cells are expected to have complex interactions with hepatocytes. However, the mechanism underlying this interaction is not yet understood. Hence, we developed cellular complexes containing a mixture of human hepatocellular carcinoma cell line (Hep-G2) and rat insulin-secreting cell line (RIN-5F) by using a co-culture model and studied the function and morphology of the resultant hybrid cellular spheroids (HCSs). The RIN-5F and Hep-G2 cells were suspension cultured and, within 5 days of culture, the two types of cells aggregated to yield spheroids. The functionality of the thus formed HCSs was evaluated by measuring the levels of insulin and albumin in the culture supernatant. The HCSs retained their insulin- and albumin-secreting ability and their morphology, as revealed by immunohistological staining. The insulin and albumin levels secreted by the HCSs were considerably higher than those secreted by spheroids of single-cell origin. Generally, obtaining complexes from more than two types of cells is difficult. However, we were able to generate HCSs. We believe that this culture method could have various applications such as studying the in vitro cell-cell interactions and developing new cell transplantation models. [Copyright &y& Elsevier]

Abstract: Introduction: When patients do not become insulin independent after islet cell transplantation (ICT), another aim is to eliminate severe hypoglycemia. Previously we reported that a secretory unit of islet transplant objects (SUITO) index score >10 was associated with a reduction of severe hypoglycemia. In this study, we assessed patients'' satisfaction with their insulin therapy based on the SUITO index. Methods: The study involved 11 islet recipients with type 1 diabetes who underwent ICT but still used insulin. From those patients, 41 Insulin Therapy Satisfaction Questionnaires (ITSQ) were collected. The SUITO index (fasting C-peptide [ng/mL] × 1500/blood glucose [mg/dL] − 63) was calculated at the same outpatient visits that the survey was administered. ITSQ scores were summarized using subscales and compared among 3 groups: the pre-ICT group, the low-SUITO group (SUITO index score <10 post-ICT), and the high-SUITO group (SUITO index score ≥10). Higher survey scores indicated better satisfaction. Results: Significant trend relationships across the 3 groups were observed in the ITSQ total score (P = .02 with Jonckheere-Terpstra test) and subscale scores of glycemic control (P < .001), hypoglycemic control (P = .01), and inconvenience of regimen (P = .004). The pairwise comparisons between the 3 groups found significant differences: high SUITO versus both pre-ICT and low SUITO for the total ITSQ score (P = .03 and .005, respectively) and glycemic control score (P = .008 and .001, respectively), and high SUITO versus low SUITO for hypoglycemic control score (P = .04) and inconvenience of regimen score (P = .008). Conclusion: Islet recipients with a SUITO index ≥10 experienced higher satisfaction with insulin injection therapy compared with the pre-ICT group, even though they were insulin dependent. A SUITO index ≥10 is a reasonable benchmark for successful ICT. [Copyright &y& Elsevier]

Abstract: We used in situ perfusion and a multiple-organ harvesting technique to collect islets from adult pig pancreata. The tissues were digested with collagenase P followed by purification in a lympholyte discontinuous gradient using a COBE2991 cell separator. The yield and purity of isolated islets were evaluated with a light microscope after dithizone (DTZ) staining. Islet function was assessed using an in vitro insulin release assay. The results showed that before purification 275,000 ± 20,895 islet equivalents (IEQ) were obtained from 1 digested pancreas. After purification with gradient centrifugation, the islet yield was 230,350 ± 26,679 IEQ/pancreas. Each gram of the purified pancreatic tissues yielded 2710 ± 229 IEQ with an average purity of 50.2 ± 2.0%. The purified islet cells responded to stimulation with high glucose concentrations (16.7 mmol/L), namely, 4.74-fold greater than the insulin secretion with exposure to the basal level of glucose (3.3 mmol/L; P < .001). These results suggested that the established isolation method can be applied to large-scale purification of fully functional islets from pig pancreata. [Copyright &y& Elsevier]

Abstract: This work sought to summarize the main issues of the last decade in the field of clinical islet transplantation. Ten years ago in Edmonton, a new protocol initiated for islet transplantation brought a breakthrough to the field. The earlier, rather poor results were in a sharp contrast to the first published results of 100% insulin freedom at 1 year. However, later it became clear that the promising initial results decline with time; at around 5 years, only about 10% of the patients maintain freedom from external insulin. Despite that fact, a milestone was set and intensive research started worldwide. New hopes were raised for patients. Modifications of the original protocol have been implemented to improve clinical results; however, islet transplantation remains an experimental procedure to date. [Copyright &y& Elsevier]

Abstract: Introduction: New-onset diabetes mellitus, which occurs after kidney transplant and type 2 diabetes mellitus (T2DM), shares common risk factors and antecedents in impaired insulin secretion and action. Several genetic polymorphisms have been shown to be associated with T2DM. We hypothesized that transplant recipients who carry risk alleles for T2DM are “tipped over” to develop diabetes mellitus in the posttransplant milieu. Methods: We investigated the association of genetic and traditional risk factors present before transplantation and the development of new-onset diabetes mellitus after kidney transplantation (NODAT). Markers in 8 known T2DM-linked genes were genotyped using either the iPLEX assay or allelic discrimination (AD)-PCR in the study cohort testing for association with NODAT. We used univariate and multivariate logistic regression models for the association of pretransplant nongenetic and genetic variables with the development of NODAT. Results: The study cohort included 91 kidney transplant recipients with at least 1 year posttransplant follow-up, including 22 who developed NODAT. We observed that increased age, family history of T2DM, pretransplant obesity, and triglyceridemia were associated with NODAT development. In addition, we observed positive trends, although statistically not significant, for association between T2DM-associated genes and NODAT. Conclusions: These findings demonstrated an increased NODAT risk among patient with a positive family history for T2DM, which, in conjunction with the observed positive predictive trends of known T2DM-associated genetic polymorphisms with NODAT, was suggestive of a genetic predisposition to NODAT. [Copyright &y& Elsevier]

Abstract: Pancreatic islet transplantation is a promising therapeutic treatment for type 1 diabetes mellitus. In the present study, we cocultured islets with or without a polyglycolic acid (PGA) fibrous scaffold for 5 days and transplanted the PGA-islet grafts into the leg muscles of Wistar rats with streptozotocin-induced diabetes; controls were injected with saline. The results showed that the blood glucose concentrations of the group given islets embedded with the PGA scaffold were lower than those without the scaffold or controls. On the other hand, the insulin content of the PGA-islet group was higher at all 5 time points compared with the insulin contents of the other 2 groups. After transplantation, many islets in the PGA-islet grafts showed normal morphology (as seen under the scanning electron microscope) and were surrounded by red blood cells. A fibrous extracellular matrix was visible around the PGA-islet grafts. These results demonstrated that PGA-islet grafts improved blood glucose and insulin concentrations in rats with induced diabetes. [Copyright &y& Elsevier]

Abstract: Objective: Insulin is one factor responsible for hepatotrophic regeneration in animal models. This study assessed the clinical effects of intraportal administration of insulin on liver graft regeneration in adult patients undergoing right lobe living donor liver transplantation (LDLT). Methods: Between July 2005 and September 2007, 19 right lobe LDLT adult recipients voluntarily received posttransplant intraportal insulin administration. The present study describes 15 patients without postoperative vascular and bile duct complications, with more than 1 month survival and with complete clinical data who were enrolled to receive intraportal insulin therapy (group I; n = 15). Another consecutive 15 right lobe LDLT adult recipients without any stimulation regeneration who met the same criteria were enrolled in as noninsulin therapy control group (group NI; n = 15). Group I recipients were treated postoperatively with intraportal insulin infusion, as follows. An 18-gauge catheter was inserted into right gastro-omental vein during surgery, to administer regular insulin just after the operation at the rate of 2 U/h for 1 week. Graft volume (GV) was measured by computed tomography on postoperative days (POD) 7 and 30. Liver functions and serum insulin levels were also measured at POD 7 and POD 30. The liver graft regeneration rate was defined as ratio of posttransplant GV/harvested GV and posttransplant graft-to-recipient weight ratio (GRWR)/operative GRWR. Results: The rate defined as ratio of POD 7 GV/harvested GV among group I was significantly greater than that of group NI (186.07 ± 35.40% vs 160.61 ± 22.11%; P < .05). The rate defined as ratio of POD 7 GRWR/operation GRWR was also significantly higher in group I than group NI (178.95 ± 35.84% vs 156.56 ± 18.53%; P < .05), whereas there was no significant difference in terms of regeneration rates at 1 month post-LDLT. Intraportal insulin administration may significantly downregulate POD 7 total bilirubin, aspartate aminotransferase, and alanine aminotransferase levels (P < .05). These results suggested that intraportal insulin administration augmented liver regeneration during the first postoperative week by improving hepatic function in LDLT recipients. [Copyright &y& Elsevier]

Abstract: Objectives: The diabetic rhesus monkey seems to be a useful model for preclinical investigations of islet transplantation and new drug treatments for type 1 diabetes mellitus (T1DM). Information is limited regarding a standard technique to induce and assess diabetes in rhesus monkeys as well as the strategy to apply insulin administration. Herein, we have established and characterized a model of diabetic rhesus macaques. Methods: Four monkeys were divided into 2 groups of 2 each: group 1, total pancreatectomy; and group 2, partial pancreatectomy (75%) with low-dose streptozotocin (STZ) administration. Pancreatic function was measured using intravenous glucose tolerance tests before the operation. Spiral computed tomography (CT) scans of the pancreas were obtained before and after pancreatectomy. Fasting blood glucose and postprandial blood glucose levels were monitored twice daily using blood samples from the fingers or toes. Various types and doses of insulin were administered twice daily. We performed regular assessments of hematological and serum biochemical parameters, insulin, and C-peptide. Results: Both total pancreatectomy and partial pancreatectomy (75%) with STZ administration induced T1DM in rhesus monkeys; there was interindividual variation in the STZ dose. Excluding C-peptide and insulin, the hematological and serum biochemical parameters did not differ significantly from normal values postoperatively. The various insulin treatment strategies are achieved stable blood glucose (BG) levels. Conclusions: STZ injection after partial pancreatectomy may be a safe, reproducible method to induce T1DM. Porcine insulin administration was a safe, economical method to control BG levels in a diabetic rhesus monkey before islet transplantation. [Copyright &y& Elsevier]

Abstract: Assessing islet cellular composition and β cell viability using Flow Cytometry (FC) and Laser Scanning Cytometry (LSC) may aid in determining the transplant quality of islets. Human islets (2500 IEQ, n = 44, purity ≥80%) dissociated into a single cell suspension were stained with ductal marker CA19, with Newport Green (NG) and FluoZin3 (FL3) for β-cell identification, with TMRE to assess mitochondrial membrane potential, with DAPI to identify live vs. dead cells, and with Annexin-V/DAPI to differentiate apoptotic and necrotic cells. For LSC, cell preparations (n = 9) were stained for insulin (β-cells), glucagon (α-cells), somatostatin (δ cells), and pancreatic polypeptide (ppp cells). Fluorescence microscopy (EtBr/FDA) and insulin response were also measured. DAPI− staining was 73.78% ± 1.37, while EtBr/FDA was 96% ± 0.48. 52.5% ± 3.73 of all cells were NG+, of which 58.08% ± 2.61 were NG+/TMRE+. Annexin-V/DAPI staining (n = 26) showed 13.8% ± 0.89 apoptotic, 27.2% ± 2.0 necrotic, and 51.9% ± 2.22 live cells. 26.0% ± 5.19 of cells were CA19 positive (n = 17), of which 45.5% ± 4.37 were also TMRE+, and 5.2% ± 1.2 of the TMRE+ were also NG+/CA19+. NG and FL3 showed similar staining (n = 8). Comparison of short-term (≤2 days) versus long-term (≥3 days) culture showed similar TMRE+/NG+ averages, albeit lower percentages of live (36.4% vs 51.9%), and higher percentages of apoptotic (19.2% vs 13.8%) and necrotic cells (37.4% vs 27.2%) for long-term, as determined by Annexin-V staining. LSC resulted in 54.17% ± 4.62 β-cells, 33.33% ± 4.16 α-cells, 8.75% ± 2.5 δ-cells, and 3.75% ± 0.79 ppp cells. There is no significant difference between insulin positive cells and NG positive cells (P ≤ .55). FC and LSC provide valuable information about islet quality, which could potentially be used for evaluating islets prior to transplantation. [Copyright &y& Elsevier]

Abstract: Background: Extracellular microenvironment and intrinsic genetic programs determine the fate of stem cells. We observed whether mesenchymal stem cells (MSCs) contributed to insulin-producing cells in a manipulated microenvironment. Methods: We delivered pancreatic pieces into Niobium-Coated Dynamatrix to construct a simulated pancreatic microenvironment, upon which soluble cytokine exchange and direct cell–cell contact between MSCs and pancreatic cells could occur. Bone marrow–derived MSCs were cultured upon the microenvironment. Differentiated isletlike cells were observed under an inverted microscope. Insulin in supernates was measurement by enzyme-linked immunosorbent assay. Insulin and c-peptide expression were verified by fluorescent immunocytochemistry and fluorescence in situ hybridization. Apoptosis of isletlike masses in high-glucose DMEM was detected by FACS. Results: After 3 to 4 weeks in culture, typical isletlike masses were observed. Insulin secreted by differentiated cells (414.47 ± 30.30 mIU/L) was much greater than that of undifferentiated cells (4.89 ± 1.01 mIU/L; P < .05). Insulin and c-peptide expression were positive both in protein and mRNA levels. The transdifferentiated isletlike mass did not undergo apoptosis after another 3 weeks of culture in high-glucose DMEM. Conclusion: This simulated injury microenvironment without induction guided MSCs to functional isletlike cells hopefully to replace β cells. [Copyright &y& Elsevier]

Abstract: Our objective was to investigate the potential protective effects of insulin on the liver injury induced in three ischemia and reperfusion (I/R) models. Methods: Three I/R models were used: (1) I/R of the liver was produced in isolated, perfused rat livers; (2) in in situ I/R of the liver in rats, ischemia was induced by clamping off the hepatic artery and portal vein for 40 minutes, the flow then restored, and the liver reperfused for 90 minutes; (3) in in situ I/R of the liver in mice, ischemia was induced by clamping off the hepatic artery for 15 minutes, the flow then restored, and the liver reperfused for 45 minutes. In all three cases, blood samples collected before ischemia and after reperfusion were analyzed for sGOT. Plasma nitrate/nitrite, hydroxyl radicals, and tumor necrosis factor were also measured. In each model, a dose of insulin sufficient to induce euglycemia was administered to assess its protective effect on liver injury and inflammation. Results: These I/R protocols resulted in a significant increase in sGOT and in three inflammatory parameters; nitric oxide, hydroxyl radicals, and tumor necrosis factor. Pretreatment with insulin did not attenuate the liver injury in any of the three I/R models. Conclusions: Although insulin has been reported to provide anti-inflammatory benefits by reducing oxidative and nitrosative stress and cytokine release, none of these protective effects was seen in the three I/R-induced liver injury models we tested. [Copyright &y& Elsevier]

Abstract: Diabetes mellitus appearing after kidney transplantation—posttransplant diabetes mellitus (PTDM)—is a common complication associated with poor graft and patient survival. The purpose of the current study was to determine the risk factors for developing PTDM in 204 renal transplant recipients who had been followed for at least 30 months. Posttransplant diabetes mellitus was diagnosed according to the American Diabetic Association/WHO criteria, or a requirement for insulin, an oral hypoglycemic agent, or both. Analyses of possible risk factors for PTDM included demographic features, dialysis and posttransplantation duration, smoking, body mass index, medications, co-morbid diseases, HLA mismatches, as well as laboratory metrics of serum creatinine, albumin, calcium, phosphorus, C-reactive protein, parathyroid hormone, and lipid profiles. Twenty-six patients displayed PTDM. Univariate analysis showed that older age, greater body mass index, presence of hepatitis C virus (HCV) infection, and smoking at the time of renal transplantation were associated with PTDM development. In a multivariate analysis, HCV infection, smoking, and patient age at the time of transplantation were independent risk factors for PTDM. In conclusion, the presence of HCV infection or a smoking habit in addition to older age at the time of transplantation were the main predictors for developing PTDM. Patients should be closely followed regarding their smoking habit and weight gain as modifiable risk factors for PTDM. [Copyright &y& Elsevier]

Abstract: The aim of this study was to preliminarily evaluate the duodenal patch technique combined with open sphincterotomy in terms of prevention of graft pancreatitis. From April 2003 to March 2005, 17 simultaneous pancreas and kidney transplantations were performed using this technique. All recipients are alive with good renal transplant function. Directly after sphincterotomy in 16 pancreatic grafts a good outflow of clear pancreatic juice and a lessening of graft tenseness were observed during surgery. In two transplants an additional sphincterotomy of the Santorini duct sphincter was necessary. In one recipient no pancreatic juice secretion was observed and insulin independence was not obtained. This graft was explanted shortly afterward. In 13 recipients no graft pancreatic or peripancreatic fluid collection requiring intervention was observed. Of the three recipients who developed graft pancreatitis, two required graft pancreatectomy. In conclusion, Sphincterotomy facilitates pancreatic juice outflow by reducing intraoperative graft edema, which could lead to subsequent inflammation. Further studies on the factors inducing graft pancreatitis are necessary to eliminate this severe complication. [Copyright &y& Elsevier]

Abstract: Rapid reestablishment of a functional microvasculature in transplanted islets is crucial for islet survival and function. To illustrate the importance of angiogenesis in islet engraftment, we took a loss-of-function approach to block angiogenesis in newly transplanted islets and determined the extent of islet engraftment in correlation with islet mass and glycemic control in diabetic recipient mice. Diabetic mice were transplanted with a marginal mass of 200 islets under the renal capsule, followed by once-daily oral administration of saline or 150 mg/kg of C-statin, a potent angiogenic inhibitor, for 14 days. Blood glucose profiles and the amplitude of glucose-stimulated insulin secretion in engrafted islets were determined. At 30 days posttransplant, islet grafts were retrieved for the determination of insulin content and vascular density by immunohistochemistry. When compared to sham-treated controls, diabetic recipient mice receiving a daily oral dose of C-statin exhibited significantly impaired blood glucose profiles and diminished glucose-stimulated insulin secretion in response to glucose challenge, correlating with significantly reduced intragraft insulin content and vascular density. Selective inhibition of angiogenesis was associated with reduced islet mass in diabetic mice. These data extend our view that rapid onset of angiogenesis is crucial for islet survival and engraftment and support the development of therapeutic strategies to stimulate angiogenesis in newly implanted islets for enhancing islet engraftment and improving the outcome of marginal islet transplantation. [Copyright &y& Elsevier]

Abstract: Short-term stimulation with insulinotropic factors may induce morphologic and functional changes in primary ductal cell cultures as a potential source of stem cells. We sought to assess the capacity of hepatocyte growth factor (HGF) to induce expression and maturation of proteins—PDX-1 and GLUT-2—and the subsequent β-cell secretory profiles. HGF, which is involved in pancreatic development, may induce islet β-cell neogenesis. Primary ductal cell monolayers were cultured in Click’s + FBS 10% at 37°C until tissue confluence. The medium was enriched with HGF (10 ng/mL for different periods); controls were treated for similar times with normal culture medium. At the end of the study, three-dimensional islet-like cell aggregates were observed in both conditions. In all conditions immunostaining studies showed positivity for the major endocrine-phenotype cell markers: insulin, PDX-1, glucokinase, and GLUT-2. Furthermore, treatment with HGF for short periods induced the expression of a functionally active, phosphorylated isoform of PDX-1. Finally, we observed that under basal conditions the cells initially and progressively released proinsulin throughout 5 days in all settings. Thereafter proinsulin was gradually replaced by insulin in the culture medium, reflecting a maturation progress. This pattern of insulin maturation and release was more evident when the cells were continuously stimulated with HGF for 12 days. The employed stimuli seemed to differentiate the original ductal cell layers toward endocrine cell phenotypes that synthesize and release proinsulin and subsequently insulin. HGF seems to provide a more efficient differentiation. [Copyright &y& Elsevier]

Abstract: In liver transplantation alterations of glucose metabolism are common but not well understood. Influence of immunosuppression is widely presumed but has not proven until now. Using a frequently sampled intravenous glucose tolerance test with a minimal modeling technique of glucose disappearance we analyzed insulin sensitivity (SI) and β-cell function (first and second phase of pancreatic β-cell secretion, Phi 1 and Phi 2) in living donor liver transplantation of the right lobe. Initial immunosuppression in recipients was done with tacrolimus, prednisolone, and basiliximab induction. Donors and recipients were investigated before and 10 days, 6 months, and 1 year after operation. Normal SI of controls (donors before operation) decreased markedly 10 days after right lobectomy to SI 2.22 ± 0.35 × 10−4 min−1 × μU/mL (P &#60; .001); Phi 2 was compensatory increased. All parameters normalized within 1 year. Recipients were insulin-resistant with hyperinsulinemia before transplantation. After transplantation no parameter was significantly different from donors; all normalized equally to donors over 1-year follow-up. Thus, immunosuppression in recipients has no influence on glucose metabolism because liver function itself seems to play a more pronounced role than known until now. [Copyright &y& Elsevier]

Abstract: The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) established the Collaborative Islet Transplant Registry (CITR) in September 2001. In its inaugural year, CITR represents the efforts of 12 collaborating North American islet transplant centers reporting 86 islet transplant recipients'' data (1999¿2003) and 173 processed pancreata leading to 158 infusion procedures. Recipient median age was 42.2 years, duration of diabetes was 30 years, and over 66% of the recipients were female. Twenty-eight patients received a total of 1 islet infusion, 44 patients received 2 islet infusions, and 14 patients received 3 islet infusions. The median age of the deceased donor was 44 years and body mass index was 28.2. Median time from cross clamp to pancreas recovery was 27 minutes, while duration of cold ischemia was 7 hours. Over 77% of the processing facilities used a density gradient for islet purification. For recipients of only 1 infusion, approximately 8665 total islet equivalents/kg were infused; recipients of 2 infusions received 14,102 islet equivalents/kg, and recipients of 3 infusions received 22,922 islet equivalents/kg. At 6 months after the last infusion, 61.1% of the recipients were reported to be insulin independent; at 12 months, 57.9% were reported to be insulin independent. There have been no deaths reported to CITR; 45 serious adverse events were reported. Through its collaboration with the islet transplant community and its interaction with professional societies and federal agencies, CITR is positioned to provide current and comprehensive information on clinically significant outcome measures in islet transplantation. [Copyright &y& Elsevier]

Abstract: Previous studies suggest that multiple transplantations might be equally efficient to a single regimen for human adult islets. The aim of this study was to compare metabolic parameters after each of the two regimens of human fetal islet (HFI) transplantation in type 1 diabetics. In group A (single transplant, n = 9), 180 ± 20 × 1000 HFI equivalents (IEQs) were implanted by a single IM injection; in group B (multiple transplants, n = 8) islets were implanted as three consecutive injections (60 ± 10 × 1000 IEQs) at 7-day intervals. We analyzed the metabolic parameters on days −1, 30, 60, 90, 120, 150, and 180 after the procedure. Among the metabolic parameters, we evaluated insulin secretion capacity-ISC (C peptide, RIA), metabolic control (HbA1c, chromatography), and insulin daily dose IDD. We found that C peptide levels increased, peaking on day 90 (A: 0.38 ± 0.15; B: 0.34 ± 0.19 nmol/L, P = NS) and then rapidly decreasing without differences, the HbA1c levels and IDD decreased in the same manner without differences between the groups. Our results demonstrate that multiple and single islet transplant regimens are equally efficient to temporarily restore a significant ISC with improvement of metabolic and clinical parameters. The results imply that the two regimens have an equal clinical value. [Copyright &y& Elsevier]

Abstract: Introduction: According to previous estimates from large animals and man, a minimum of approximately 5000 to 6000 engrafted islet equivalents (IEQ)/kg recipient weight is critical to establish insulin independence. Utilizing a single donor, this threshold yield of purified islets can be retrieved from approximately one third of all isolations. The aim of this study was to improve human islet purification by optimization of the osmolality and the density range of the continuous Ficoll-sodium-diatrizoate (FSD) gradient to facilitate consistent purities >80% of human islet preparations without considerable loss of islet yield. Methods: Aliquots of human pancreatic digests were placed on continuous density gradients. After centrifugation, sequential aliquots were extracted for amylase and insulin to determine the relative and cumulative density distribution of endocrine and exocrine tissue. We addressed the impact of two factors: (1) osmolalities (300 to 600 mosm/kg) in the gradient of FSD covering a density range of 1.070 to 1.100 g/cm3; and (2) density (FSD 500/1.070 to 1.100) versus density-osmolarity gradient (DO-FSD 400–530/1.080 to 1.113). Results: The density of exocrine and endocrine tissue increased with rising osmolality. Differences in density of both tissues were highest at 450 and lowest at 300 and 600 mOsmol/kg. Purity and recovery were highest at 450 versus 400 or 500 mOsm/kg (NS). Exocrine but not endocrine tissue was more dense in DO-FSD than in FSD gradient (P < .05). The differences in density were 0.004 versus 0.013 g/cm3 (P < .01), resulting in an increased islet purity and recovery. Conclusion: The best osmolality for the FSD 1.070 to 1.100 g/cm3 is at 450 mOsm/kg. Using the DO-FSD may improve human islet purification allowing successful clinical islet transplantation. [Copyright &y& Elsevier]