Nutrition support in the pediatric total pancreatectomy with islet autotransplantation recipient.

For children with diminished quality of life and chronic pain caused by acute recurrent or chronic pancreatitis who are undergoing total pancreatectomy with islet autotransplantation, postoperative nutrition support has several unique characteristics. Surgical complications may lead to delays in nutrition support initiation or require modifications to the regimen. Early postoperative dysmotility requires the use of temporary enteral nutrition until this improves. The resultant complete exocrine pancreatic insufficiency necessitates lifelong pancreatic enzyme replacement therapy and fat-soluble vitamin supplementation. A low-oxalate diet is recommended to prevent kidney stones. Carbohydrate counting is needed for the provision of short-term insulin dosing and possibly long-term as well, depending on the transplanted islet yield. Children should have careful nutrition assessment and monitoring at several follow-up visits during the first year, then annually, and at any time with concerns.

Decellularized human pancreatic extracellular matrix-based physiomimetic microenvironment for human islet culture.

A strategy that seeks to combine the biophysical properties of inert encapsulation materials like alginate with the biochemical niche provided by pancreatic extracellular matrix (ECM)-derived biomaterials, could provide a physiomimetic pancreatic microenvironment for maintaining long-term islet viability and function in culture. Herein, we have demonstrated that incorporating human pancreatic decellularized ECM within alginate microcapsules results in a significant increase in Glucose Stimulation Index (GSI) and total insulin secreted by encapsulated human islets, compared to free islets and islets encapsulated in only alginate. ECM supplementation also resulted in long-term (58 days) maintenance of GSI levels, similar to that observed in free islets at the first time point (day 5). At early time points in culture, ECM promoted gene expression changes through ECM- and cell adhesion-mediated pathways, while it demonstrated a mitochondria-protective effect in the long-term. STATEMENT OF SIGNIFICANCE: The islet isolation process can damage the islet extracellular matrix, resulting in loss of viability and function. We have recently developed a detergent-free, DI-water based method for decellularization of human pancreas to produce a potent solubilized ECM. This ECM was added to alginate for microencapsulation of human islets, which resulted in significantly higher stimulation index and total insulin production, compared to only alginate capsules and free islets, over long-term culture. Using ECM to preserve islet health and function can improve transplantation outcomes, as well as provide novel materials and platforms for studying islet biology in microfluidic, organ-on-a-chip, bioreactor and 3D bioprinted systems.

Nutritional Risks in Patients Undergoing Total Pancreatectomy Islet AutoTransplantation in the POST Consortium.

BACKGROUND AND AIMS: Total pancreatectomy with islet autotransplantation (TPIAT) can relieve pain for individuals with acute recurrent or chronic pancreatitis. However, TPIAT may increase the risk of poor nutritional status with complete exocrine pancreatic insufficiency, partial duodenectomy, and intestinal reconstruction. Our study's objective was to evaluate nutritional status, anthropometrics, and vitamin levels before and after TPIAT. METHODS: The multicenter Prospective Observational Study of TPIAT (POST) collects measures including vitamins A, D, and E levels, pancreatic enzyme dose, and multivitamin (MVI) administration before and 1-year after TPIAT. Using these data, we studied nutritional and vitamin status before and after TPIAT. RESULTS: 348 TPIAT recipients were included (68% adult, 37% male, 93% Caucasian). In paired analyses at 1-year follow-up, vitamin A was low in 23% (vs 9% pre-TPIAT, p < 0.001); vitamin E was low in 11% (vs 5% pre-TPIAT, p = 0.066), and 19% had vitamin D deficiency (vs 12% pre-TPIAT, p = 0.035). Taking a fat-soluble multivitamin (pancreatic MVI) was associated with lower risk for vitamin D deficiency (p = 0.002). Adults were less likely to be on a pancreatic MVI at follow-up (34% vs 66% respectively, p < 0.001). Enzyme dosing was adequate. More adults versus children were overweight or underweight pre- and post-TPIAT. Underweight status was associated with vitamin A (p = 0.014) and E (p = 0.02) deficiency at follow-up. CONCLUSIONS: Prevalence of fat-soluble vitamin deficiencies increased after TPIAT, especially if underweight. We strongly advocate that all TPIAT recipients have close post-operative nutritional monitoring, including vitamin levels. Pancreatic MVIs should be given to minimize risk of developing deficiencies.

Investigation of the effect of pancreatic decellularized matrix on encapsulated Islets of Langerhans with mesenchymal stem cells.

OBJECTIVE: In this study, it was aimed to provide a therapeutic approach for T1DM by encapsulating the pancreatic islets with mesenchymal stem cells and decellularized pancreatic extracellular matrix to support the survival of islets while maintaining their cellular activity. METHOD: Pancreatic extracellular matrix was decellularized using different concentrations of detergent series. After the preparation of the protein-based tissue extracellular matrix was shown to be free of cells or any genetic material by molecular, immunofluorescence and histochemical techniques. Following the homogenization of the decellularized pancreatic extracellular matrix and the analysis of its protein composition by LC-MS, the matrix proteins were incorporated with pancreatic islets and rat adipose tissue-derived MSCs (rAT-MSCs) in alginate microcapsules. Glucose-stimulated insulin secretion property of the islet cells in the microbeads was evaluated by insulin ELISA. The gene expression profile of the encapsulated cells was analyzed by Real-Time PCR. RESULTS: Unlike the protein composition of whole pancreatic tissue, the decellularized pancreas matrix was free of histone proteins or proteins originated from mitochondria. The protein matrix derived from pancreatic tissue was shown to support the growth and maintenance of the islet cells. When compared to the non-encapsulated pancreatic islet, the encapsulated cells demonstrate to be more efficient in terms of insulin expression. CONCLUSION: The extracellular pancreatic matrix obtained in this study was directly used as supplementary in the alginate-based microcapsule enhancing the cell survival. The tissue matrix protein and alginate had a synergistic effect on total insulin secretion, which might have the potential to overcome the insulin deficiency. Despite the improvement in the cell viability and the number, the efficiency of the insulin secretion in response to glucose stimulation from the alginate microcapsules did not meet the expectation when compared with the non-encapsulated pancreatic islets.

Extracellular matrix inclusion in immunoisolating alginate-based microcapsules promotes longevity, reduces fibrosis, and supports function of islet allografts in vivo.

Immunoisolation of pancreatic-islets in alginate-microcapsules is applied to treat diabetes. However, long-term islet function is limited, which might be due to damaged and lack of contact with pancreatic extracellular matrix (ECM) components. Herein we investigated the impact of collagen IV combined with laminin sequences, either RGD, LRE, or PDSGR, on graft-survival of microencapsulated bioluminescent islets in vivo. Collagen IV with RGD had the most pronounced effect. It enhanced after 8-week implantation in immune-incompetent mice the bioluminescence of allogeneic islets by 3.2-fold, oxygen consumption rate by 14.3-fold and glucose-induced insulin release by 9.6-fold. Transcriptomics demonstrated that ECM enhanced canonical pathways involving insulin-secretion and that it suppressed pathways related to inflammation and hypoxic stress. Also, 5.8-fold fewer capsules were affected by fibrosis. In a subsequent longevity study in immune-competent mice, microencapsulated allografts containing collagen IV and RGD had a 2.4-fold higher functionality in the first week after implantation and remained at least 2.1-fold higher during the study. Islets in microcapsules containing collagen IV and RGD survived 211 ± 24.1 days while controls survived 125 ± 19.7 days. Our findings provide in vivo evidence for the efficacy of supplementing immunoisolating devices with specific ECM components to enhance functionality and longevity of islet-grafts in vivo. STATEMENT OF SIGNIFICANCE: Limitations in duration of survival of immunoisolated pancreatic islet grafts is a major obstacle for application of the technology to treat diabetes. Accumulating evidence supports that incorporation of extracellular matrix (ECM) molecules in the capsules enhances longevity of pancreatic islets. After selection of the most efficacious laminin sequence in vitro, we show in vivo that inclusion of collagen IV and RGD in alginate-based microcapsules enhances survival, insulin secretion function, and mitochondrial function. It also suppresses fibrosis by lowering proinflammatory cytokines secretion. Moreover, transcriptomic analysis shows that ECM-inclusion promotes insulin-secretion related pathways and attenuates inflammation and hypoxic stress related pathways in islets. We show that inclusion of ECM in immunoisolating devices is a promising strategy to promote long-term survival of islet-grafts.

The Effects of Photobiomodulation Therapy on Porcine Islet Insulin Secretion.

Background and objective: Xenotransplantation of porcine islets to human recipients has been investigated as a potential cure for type 1 diabetes. However, the porcine islets have poor insulin secretion capacity compared with human islets. The objective of this study was to evaluate the effect of photobiomodulation therapy (PBMT) in insulin secretion on isolated porcine islets. Methods: Eight pancreata were harvested from crossbred market porcine and the islets were isolated from the pancreas. The isolated islets were treated with PBMT (wavelength: 633 nm and dosages: 0.0, 15.6, and 31.3 J/cm2) followed by 30-min incubation in low (3.0 mM) or high (16.7 mM) glucose. The relative percentage differences on insulin secretion between three dosages were compared in low and high glucose, respectively. Results: Insulin secretion was higher in samples exposed to 15.6 J/cm2 PBMT in low glucose (p < 0.05), but not in high glucose. When evaluating sex differences, male islets had higher insulin secretion by 15.6 J/cm2 PBMT in low glucose compared with females (p < 0.05). No significant differences were seen in high glucose. When compared within the control groups (0.0 J/cm2 PBMT), the relative changes on insulin secretion in high glucose was significantly higher on male islets (p < 0.05), but not on female islets. Conclusions: PBMT may increase insulin secretion on isolated porcine islets in basal condition, but it may not improve islets' glucose responsiveness to secrete insulin. Male porcine islets may respond to PBMT and glucose stimuli better than female islets on insulin secretion.

Possibility of adiponectin use to improve islet transplantation outcomes.

Although islet transplantation (ITx) is a promising therapy for severe diabetes mellitus, further advancements are necessary. Adiponectin, an adipokine that regulates lipid and glucose metabolism, exerts favorable effects on islets, such as reinforcement of the insulin-releasing function. This study evaluated the possibility of adiponectin use to improve ITx outcomes. We treated mouse islets with 10 µg/mL recombinant mouse adiponectin by overnight culture and then assessed the insulin-releasing, angiogenic, and adhesion functions of the islets. Furthermore, 80 syngeneic islet equivalents with or without adiponectin treatment were transplanted into the renal subcapsular space of diabetic mice. In in vitro assessment, released insulin at high glucose stimulation, insulin content, and expressions of vascular endothelial growth factor and integrin ß1 were improved in adiponectin-treated islets. Furthermore, adiponectin treatment improved the therapeutic effect of ITx on blood glucose levels and promoted angiogenesis of the transplanted islets. However, the therapeutic effect was not pronounced in glucose tolerance test results. In conclusion, adiponectin treatment had preferable effects in the insulin-releasing, angiogenic, and adhesion functions of islets and contributed to the improvement of ITx. The future use of adiponectin treatment in clinical settings to improve ITx outcomes should be investigated.

Bioenergetics of Islet Preparations in a Pilot Clinical Trial of Peri-Transplant Hydroxychloroquine for Autologous Islet Transplantation.

The inflammatory response is an obstacle to success in both allogeneic and autologous islet transplantation. In autologous islet transplantation (AIT), however, the recipient is also the donor, permitting pretreatment of donor/recipient for a controlled duration prior to transplantation. We sought to exploit this feature of (AIT) by pretreating donor/recipients with chronic pancreatitis undergoing total pancreatectomy and autologous islet transplantation (TPAIT) to test the hypothesis that peri-transplant treatment with the FDA-approved anti-inflammatory hydroxychloroquine (HCQ) improves graft function. In this randomized placebo-controlled pilot clinical study, patients (n = 6) were treated with oral HCQ for 30 days prior to and 90 days after TPAIT. In vivo islet function was assessed via Mixed Meal Tolerance Testing before HCQ treatment, 6- and 12-months after surgery. In vitro islet bioenergetics were assessed at the time of transplantation via extracellular flux analysis of islet preparation samples from the clinical trial cohort and six additional patients (n = 12). Our study shows that HCQ did not alter clinical endpoints, but HCQ-treated patients showed greater spare respiratory capacity (SRC) compared to samples from control patients (P=0.028). Glycolytic metabolism of islet preparations directly correlated with stimulated C-peptide secretion both before and after TPAIT (P=0.01, R2=0.489 and P=0.03, R2=0.674, respectively), and predicted in vivo islet function better than mitochondrial metabolism of islet preps or islet equivalents infused. Overnight culture of islet preparations altered bioenergetic function, significantly decreasing SRC and maximal respiration (P<0.001). In conclusion, while HCQ did not alter clinical outcomes, it was associated with significantly increased SRC in islet preparations. Bioenergetic analyses of islet preparations suggests that culture should be avoided and that glycolysis may be a more sensitive indicator of in vivo islet function than current metrics, including islet oxygen consumption and islet equivalents infused.

Engineering a macroporous oxygen-generating scaffold for enhancing islet cell transplantation within an extrahepatic site.

Insufficient oxygenation is a serious issue arising within cell-based implants, as the hypoxic period between implantation and vascularization of the graft is largely unavoidable. In situ oxygen supplementation at the implant site should significantly mitigate hypoxia-induced cell death and dysfunction, as well as improve transplant efficacy, particularly for highly metabolically active cells such as pancreatic islets. One promising approach is the use of an oxygen generating material created through the encapsulation of calcium peroxide within polydimethylsiloxane (PDMS), termed OxySite. In this study, OxySite microbeads were incorporated within a macroporous PDMS scaffold to create a single, streamlined, oxygen generating macroporous scaffold. The resulting OxySite scaffold generated sufficient local oxygenation for up to 20 days, with nontoxic levels of reaction intermediates or by-products. The benefit of local oxygen release on transplant efficacy was investigated in a diabetic Lewis rat syngeneic transplantation model using a clinically relevant islet dosage (10,000 IEQ/kg BW) with different isolation purities (80%, 90%, and 99%). Impure islet preparations containing pancreatic non-islet cells, which are common in the clinical setting, permit examination of the effect of increased overall oxygen demand. Our transplantation outcomes showed that elevating the oxygen demand of the graft with decreasing isolation purity resulted in decreased graft efficacy for control implants, while the integration of OxySite significantly mitigated this impact and resulted in improved graft outcomes. Results highlight the superior clinical translational potential of these off-the-shelf OxySite scaffolds, where islet purity and the overall oxygen demands of implants are increased and highly variable. The oxygen-generating porous scaffold further provides a broad platform for enhancing the survival and efficacy of cellular implants for numerous other applications. STATEMENT OF SIGNIFICANCE: Hypoxia is a serious issue within tissue engineered implants. To address this challenge, we developed a distinct macroporous scaffold platform containing oxygen-generating microbeads. This oxygen-generating scaffold showed the potential to support clinically relevant cell dosages for islet transplantation, leading to improved treatment efficacy. This platform can also be used to mitigate hypoxia for other biomedical applications.

Perioperative Nutritional Aspects in Total Pancreatectomy: A Comprehensive Review of the Literature.

Total pancreatectomy (TP) is a highly invasive procedure often performed in patients affected by anorexia, malabsorption, cachexia, and malnutrition, which are risk factors for bad surgical outcome and even may cause enhanced toxicity to chemo-radiotherapy. The role of nutritional therapies and the association between nutritional aspects and the outcome of patients who have undergone TP is described in some studies. The aim of this comprehensive review is to summarize the available recent evidence about the influence of nutritional factors in TP. Preoperative nutritional and metabolic assessment, but also intra-operative and post-operative nutritional therapies and their consequences, are analyzed in order to identify the aspects that can influence the outcome of patients undergoing TP. The results of this review show that preoperative nutritional status, sarcopenia, BMI and serum albumin are prognostic factors both in TP for pancreatic cancer to support chemotherapy, prevent recurrence and prolong survival, and in TP with islet auto-transplantation for chronic pancreatitis to improve postoperative glycemic control and obtain better outcomes. When it is possible, enteral nutrition is always preferable to parenteral nutrition, with the aim to prevent or reduce cachexia. Nowadays, the nutritional consequences of TP, including diabetes control, are improved and become more manageable.

Toll-like receptor 2-modulating pectin-polymers in alginate-based microcapsules attenuate immune responses and support islet-xenograft survival.

Encapsulation of pancreatic islets in alginate-microcapsules is used to reduce or avoid the application of life-long immunosuppression in preventing rejection. Long-term graft function, however, is limited due to varying degrees of host tissue responses against the capsules. Major graft-longevity limiting responses include inflammatory responses provoked by biomaterials and islet-derived danger-associated molecular patterns (DAMPs). This paper reports on a novel strategy for engineering alginate microcapsules presenting immunomodulatory polymer pectin with varying degrees of methyl-esterification (DM) to reduce these host tissue responses. DM18-pectin/alginate microcapsules show a significant decrease of DAMP-induced Toll-Like Receptor-2 mediated immune activation in vitro, and reduce peri-capsular fibrosis in vivo in mice compared to higher DM-pectin/alginate microcapsules and conventional alginate microcapsules. By testing efficacy of DM18-pectin/alginate microcapsules in vivo, we demonstrate that low-DM pectin support long-term survival of xenotransplanted rat islets in diabetic mice. This study provides a novel strategy to attenuate host responses by creating immunomodulatory capsule surfaces that attenuate activation of specific pro-inflammatory immune receptors locally at the transplantation site.

Fat-soluble Vitamin Deficiency is Common in Children With Chronic Pancreatitis Undergoing Total Pancreatectomy With Islet Autotransplantation.

ABSTRACT: The prevalence of fat-soluble vitamin (FSV) deficiency in children undergoing total pancreatectomy with islet autotransplantation (TPIAT) for chronic pancreatitis (CP) is unknown. We quantified FSV deficiency in 100 children (age ≤18) undergoing TPIAT. FSV levels (vitamins A, E, D) and clinical history were abstracted from medical records. Vitamin A was low in 4% before and 7% at 1 year after TPIAT, vitamin E in 17% and 18%, and vitamin D in 22% and 24%, respectively, regardless of pancreatic enzyme or vitamin supplement dosing. Longer duration of CP was associated with pre-TPIAT vitamin D insufficiency (P = 0.0002). This remained significant in a multivariate regression model (adjusted P = 0.01). On multivariate analysis, there were no significant predictors of low FSV levels post-TPIAT. FSV deficiencies are common among children undergoing TPIAT and patients who have had longer disease duration may be at increased risk. All children should be monitored for FSV deficiency after TPIAT.

Combined use of GABA and sitagliptin promotes human ß-cell proliferation and reduces apoptosis.

γ-Aminobutyric acid (GABA) and glucagon-like peptide-1 receptor agonist (GLP-1RA) improve rodent ß-cell survival and function. In human ß-cells, GABA exerts stimulatory effects on proliferation and anti-apoptotic effects, whereas GLP-1RA drugs have only limited effects on proliferation. We previously demonstrated that GABA and sitagliptin (Sita), a dipeptidyl peptidase-4 inhibitor which increases endogenous GLP-1 levels, mediated a synergistic ß-cell protective effect in mice islets. However, it remains unclear whether this combination has similar effects on human ß-cell. To address this question, we transplanted a suboptimal mass of human islets into immunodeficient NOD-scid-gamma mice with streptozotocin-induced diabetes, and then treated them with GABA, Sita, or both. The oral administration of either GABA or Sita ameliorated blood glucose levels, increased transplanted human ß-cell counts and plasma human insulin levels. Importantly, the combined administration of the drugs generated significantly superior results in all these responses, as compared to the monotherapy with either one of them. The proliferation and/or regeneration, improved by the combination, were demonstrated by increased Ki67+, PDX-1+, or Nkx6.1+ ß-cell numbers. Protection against apoptosis was also significantly improved by the drug combination. The expression level of α-Klotho, a protein with protective and stimulatory effects on ß cells, was also augmented. Our study indicates that combined use of GABA and Sita produced greater therapeutic benefits, which are likely due to an enhancement of ß-cell proliferation and a decrease in apoptosis.

Diazoxide Preconditioning of Nonhuman Primate Pancreas Improves Islet Isolation Outcomes by Mitochondrial Protection.

OBJECTIVES: Previously, we showed that diazoxide (DZ), an effective ischemic preconditioning agent, protected rodent pancreas against ischemia-reperfusion injury. Here, we further investigate whether DZ supplementation to University of Wisconsin (UW) solution during pancreas procurement and islet isolation has similar cytoprotection in a preclinical nonhuman primate model. METHODS: Cynomolgus monkey pancreata were flushed with UW or UW + 150 µM DZ during procurement and preserved for 8 hours before islet isolation. RESULTS: First, a significantly higher islet yield was observed in UW + DZ than in UW (57,887 vs 23,574 IEq/pancreas and 5396 vs 1646 IEq/g). Second, the DZ treated islets had significantly lower apoptotic cells per islet (1.64% vs 9.85%). Third, DZ significantly inhibited ROS surge during reperfusion with a dose-response manner. Fourth, DZ improved in vitro function of isolated islets determined by mitochondrial potentials and calcium influx in responses to glucose and KCI. Fifth, the DZ treated islets had much higher cure rate and better glycemia control in diabetic mice transplant model. CONCLUSIONS: This study showed a strong mitochondrial protection of DZ on nonhuman primate islets against ischemia-reperfusion injury that provides strong evidence for its clinical application in islet and pancreas transplantation.

3-Dimensional nature-based therapeutics in pediatric patients with total pancreatectomy and islet auto-transplant.

BACKGROUND: Virtual Reality (VR) has been increasingly employed as a therapeutic means to help patients reduce stress, anxiety, and pain. While it has been shown to be effective in multiple settings, there is still scant literature referencing its use in the pediatric intensive care unit (ICU) and none using VR longitudinally as a vehicle for mindful focus utilizing natural environments. OBJECTIVES: This proof of concept study aims to demonstrate that the use of 3-D Nature-Based Therapy (NBT) glasses will lead to a reduction in pain, nausea, and anxiety in children and adolescents undergoing Total Pancreatectomy Islet Auto-Transplant (TPIAT). METHODS: Six pediatric patients (8-18 yr.) scheduled to receive TPIAT were recruited over a one-year period. Patients rated their symptoms using various scoring methods, including a novel nature-based anxiety scale. If VR was used prior to a physical therapy session, this was also noted. Patients then utilized the Oculus ™ VR device and re-scored their symptoms. Interviews were performed at entry to study, post-ICU, and at hospital discharge. RESULTS: Four of six recruited patients utilized the VR device, three of whom completed pre- and post-use scores during 11 encounters, though many other encounters occurred without scoring. Of the two patients not utilizing the device, one chose to use other means of distraction and the other reported nausea and chose not to use device. Of the patients who utilized the device, there was a net decrease in symptom scores after use, including the use of the nature-based scale which mimicked both validated scales. On survey results, all patients who utilized the device found it to be "enjoyable" and "helpful", either "would" or "might use" it again, and "would recommend it to others" for use. CONCLUSIONS: In this proof of concept study, children in a critical care setting were able to utilize VR devices for NBT after extensive surgical procedures. Initial quantitative scoring systems suggest overall improvement in symptom management, and reactions by both patients and their parents were overall positive.

Necrostatin-1 supplementation enhances young porcine islet maturation and in vitro function.

BACKGROUND: Necroptosis has been demonstrated to be a primary mechanism of islet cell death. This study evaluated whether the supplementation of necrostatin-1 (Nec-1), a potent inhibitor of necroptosis, to islet culture media could improve the recovery, maturation, and function of pre-weaned porcine islets (PPIs). METHODS: PPIs were isolated from pre-weaned Yorkshire piglets (8-15 days old) and either cultured in control islet culture media (n = 6) or supplemented with Nec-1 (100 µM, n = 5). On days 3 and 7 of culture, islets were assessed for recovery, insulin content, viability, cellular composition, GLUT2 expression in beta cells, differentiation of pancreatic endocrine progenitor cells, function, and oxygen consumption rate. RESULTS: Nec-1 supplementation induced a 2-fold increase in the insulin content of PPIs on day 7 of culture. When compared to untreated islets, Nec-1 treatment doubled the beta- and alpha-cell composition and accelerated the development of delta cells. Additionally, beta cells of Nec-1-treated islets had a significant upregulation in GLUT2 expression. The enhanced development of major endocrine cells and GLUT2 expression after Nec-1 treatment subsequently led to a significant increase in the amount of insulin secreted in response to in vitro glucose challenge. Islet recovery, viability, and oxygen consumption rate were unaffected by Nec-1. CONCLUSION: This study underlines the importance of necroptosis in islet cell death after isolation and demonstrates the novel effects of Nec-1 to increase islet insulin content, enhance pancreatic endocrine cell development, facilitate GLUT2 upregulation in beta cells, and augment insulin secretion. Nec-1 supplementation to culture media significantly improves islet quality prior to xenotransplantation.

The Role of Vitamin D and Omega-3 PUFAs in Islet Transplantation.

Recurrence of autoimmunity and allograft rejection represent major challenges that impact the success of islet transplantation. Despite the remarkable improvements achieved in immunosuppression strategies after the publication of the Edmonton protocol, long-term data of intra-hepatic islet transplantation show a gradual decline in beta-cell function. Therefore, there is a growing interest in the investigation of novel, safe and effective anti-inflammatory and immunomodulatory strategies able to promote long-term islet graft survival and notable improvements in clinical outcomes of islet transplant recipients. Vitamin D has been shown to exert anti-inflammatory and immunomodulatory effects. Pre-clinical studies investigating the use of vitamin D and its analogs (alone or in combination with immunosuppressive agents and/or other anti-inflammatory agents, such as omega-3 polyunsaturated fatty acids) showed beneficial results in terms of islet graft survival and prevention of recurrence of autoimmunity/allograft rejection in animal models of syngeneic and allogeneic islet transplantation. Moreover, epidemiologic studies demonstrated that vitamin D deficiency is highly prevalent after solid organ transplantation (e.g., heart, liver or kidney transplantation). However, studies that critically assess the prevalence of vitamin D deficiency among islet transplant recipients have yet to be conducted. In addition, prospective studies aimed to address the safety and efficacy of vitamin D supplementation as an adjuvant immunomodulatory strategy in islet transplant recipients are lacking and are therefore awaited in the future.

JAK3 inhibitor-based immunosuppression in allogeneic islet transplantation in cynomolgus monkeys.

Islet transplantation is efficacious to prevent severe hypoglycemia and glycemic liability of selected patients of type 1 diabetes. However, since calcineurin inhibitor (CNI) causes ß-cell and nephrotoxicity, alternative drug(s) with similar potency and safety profile to CNI will be highly desirable. Here we tested whether JAK3 inhibitor, tofacitinib could be used instead of tacrolimus in CIT07 immunosuppression regimen in cynomolgus nonhuman primate (NHP) model. Five independent streptozotocin (STZ)-induced diabetic monkeys were transplanted with MHC-mismatched allogeneic islets and three animals were further re-transplanted upon insufficient glycemic control or early islet graft rejection. After islet transplantation, blood glucose levels were quickly stabilized and maximal islet graft survival as measured by serum C-peptide concentration was >330, 98, >134, 31, or 22 days, respectively, after transplantation (median survival day; 98 days). Cellular and humoral immune responses were efficiently suppressed by JAK3 inhibitor-based immunosuppression during the follow-up periods. Although intermittent increases of the genome copy number of cynomolgus cytomegalovirus (CMV) were detected by quantitative real-time PCR analyses, serious infections or posttransplant lymphoproliferative disease (PTLD) was not found in all animals. Taken together, we have shown that JAK3 inhibitor could be used in replacement of tacrolimus in a highly translatable NHP islet transplantation model and these results suggest that JAK3 inhibitor will be potentially incorporated in human allogeneic islet transplantation.

Calcium: A Crucial Potentiator for Efficient Enzyme Digestion of the Human Pancreas.

BACKGROUND: Effective digestive enzymes are crucial for successful islet isolation. Supplemental proteases are essential because they synergize with collagenase for effective pancreatic digestion. The activity of these enzymes is critically dependent on the presence of Ca2+ ions at a concentration of 5-10 mM. The present study aimed to determine the Ca2+ concentration during human islet isolation and to ascertain whether the addition of supplementary Ca2+ is required to maintain an optimal Ca2+ concentration during the various phases of the islet isolation process. METHODS: Human islets were isolated according to standard methods and isolation parameters. Islet quality control and the number of isolations fulfilling standard transplantation criteria were evaluated. Ca2+ was determined by using standard clinical chemistry routines. Islet isolation was performed with or without addition of supplementary Ca2+ to reach a Ca2+ of 5 mM. RESULTS: Ca2+ concentration was markedly reduced in bicarbonate-based buffers, especially if additional bicarbonate was used to adjust the pH as recommended by the Clinical Islet Transplantation Consortium. A major reduction in Ca2+ concentration was also observed during pancreatic enzyme perfusion, digestion, and harvest. Additional Ca2+ supplementation of media used for dissolving the enzymes and during digestion, perfusion, and harvest was necessary in order to obtain the concentration recommended for optimal enzyme activity and efficient liberation of a large number of islets from the human pancreas. CONCLUSIONS: Ca2+ is to a large extent consumed during clinical islet isolation, and in the absence of supplementation, the concentration fell below that recommended for optimal enzyme activity. Ca2+ supplementation of the media used during human pancreas digestion is necessary to maintain the concentration recommended for optimal enzyme activity. Addition of Ca2+ to the enzyme blend has been implemented in the standard isolation protocols in the Nordic Network for Clinical Islet Transplantation.

BMX-001, a novel redox-active metalloporphyrin, improves islet function and engraftment in a murine transplant model.

Islet transplantation has become a well-established therapy for select patients with type 1 diabetes. Viability and engraftment can be compromised by the generation of oxidative stress encountered during isolation and culture. We evaluated whether the administration of BMX-001 (MnTnBuOE-2-PyP5+ [Mn(III) meso-tetrakis-(N-b-butoxyethylpyridinium-2-yl)porphyrin]) and its earlier derivative, BMX-010 (MnTE-2-PyP [Mn(III) meso-tetrakis-(N-methylpyridinium-2-yl)porphyrin]) could improve islet function and engraftment outcomes. Long-term culture of human islets with BMX-001, but not BMX-010, exhibited preserved in vitro viability. Murine islets isolated and cultured for 24 hours with 34 µmol/L BMX-001 exhibited improved insulin secretion (n = 3 isolations, P < .05) in response to glucose relative to control islets. In addition, 34 µmol/L BMX-001-supplemented murine islets exhibited significantly reduced apoptosis as indicated by terminal deoxynucleotidyl transferase dUTP nick end labeling, compared with nontreated control islets (P < .05). Murine syngeneic islets transplanted under the kidney capsule at a marginal dose of 150 islets revealed 58% of 34 µmol/L BMX-001-treated islet recipients became euglycemic (n = 11 of 19) compared with 19% of nontreated control islet recipients (n = 3 of 19, P < .05). Of murine recipients receiving a marginal dose of human islets cultured with 34 µmol/L BMX-001, 92% (n = 12 of 13) achieved euglycemia compared with 57% of control recipients (n = 8 of 14, P = .11). These results demonstrate that the administration of BMX-001 enhances in vitro viability and augments murine marginal islet mass engraftment.