Islet Transplantation Procedure Research Articles

In Vitro Human Fetal Pancreatic Islets to Redefine Pancreatic Research.

In vitro studies with human fetal islets of different gestational ages (GA) would be a great tool to generate information on the developmental process of the islets as this would help to recontextualize diabetes research and clinical practice. Pancreatic islets from human cadavers and other animal species are extensively researched to explore their suitability for islet transplantation procedure, one of the upcoming treatment strategies for insulin-dependent diabetes mellitus. Although human fetal islets are also considered for islet transplantation, ethical issues and limited knowledge constraints their use. The fetal islets could be explored to address the information lacunae on the maturity process of pancreatic islets and the endocrine-exocrine signaling mechanisms. This study aimed to assess the feasibility of isolating viable islets and study the cytoarchitecture of the fetal pancreas of GA22-29 weeks, not reported otherwise. Pancreas obtained from the aborted fetuses of GA 22-29 weeks were subjected to collagenase digestion and were further cultured to determine the viability in vitro. Parameters assessed were expression of markers for endocrine cell lineages and insulin release to glucose challenge. Islets were viable in vitro and islets were shown to maintain cues for post-digestion re-aggregation and expansion in culture. The immunofluorescent staining showed islets of varying sizes, homogenous cell clusters aggregating to form heterogenous cell clusters, otherwise not reported for this GA. On stimulation with different concentrations of glucose (2.8 and 28 mM), the fetal islets in the culture exhibited insulin release, and this response confirmed their viability in vitro. Our findings showed that viable islets could be isolated and cultured in vitro for further in-depth studies to explore their proliferative potential as well as for the identification of pancreatic progenitors, a good strategy to take forward.

P.101: Safety and Efficacy of a Percutaneous Intraportal Islet Transplantation Procedure Using Tract Embolization With Gelatine Foam Pledgets

Introduction: Intraportal pancreatic islet transplantation is a minimally invasive procedure to treat patients with diabetes mellitus and unstable glycemic control despite optimal medical treatment. We analysed the safety and efficacy of percutaneous transhepatic intraportal islet transplantation procedures using embolization of the hepatic puncture tract with gelatin foam pledgets. Methods: This was a retrospective, single center study including all allogeneic islet transplantations between 2007 and 2019 at the Leiden University Medical Center. Under local anaesthesia, islets were infused into the main portal vein using a right-sided transhepatic approach. Tract embolization was performed in 51/62 procedures using the Hunter Biopsy Sealing Device. Complications were assessed according to CTCAEv5.0. Stimulated C-peptide during a mixed meal test was measured after 3 months to evaluate efficacy. Results: A total of 62 islet transplants in 38 recipients (female 39.5%; median age 53.2 (IQR 40.8 – 64.0) years) were included. 11 complications occurred in 62 procedures: no CTCAE grade 4 or 5 complications occurred. Bleeding occurred in 3/11 (27.3%) of procedures without tract embolization, compared to 2/51 (3.9%) with tract embolization (p=0.01). 3 of these bleeding episodes comprised grade 3 complications which could be managed conservatively, and 2 bleeding episodes were grade 1 that did not require intervention. More than one puncture attempt was a positive predictor for bleeding (p=0.001) and pain (p=0.002). No portal vein thromboses were encountered.1 biliary puncture (grade 1) and 5 episodes of pain (grade 1 (n=3) and grade 2 (n=2)) occurred. Three months after infusion, stimulated C-peptide was >0.17 nmol/L in 60/62 (97%) transplant recipients. Conclusion: A percutaneous transhepatic procedure using gelatine foam pledgets is a safe and efficacious method to transplant islets into the portal vein. Complications were either self-limiting or could be managed with non-invasive treatment. The vast majority of patients gained endogenous insulin production.

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

Inguinal Subcutaneous White Adipose Tissue (ISWAT) Transplantation Model of Murine Islets.

Pancreatic islet transplantation is a well-established therapeutic treatment for type 1 diabetes. The kidney capsule is the most commonly used site for islet transplantation in rodent models. However, the tight kidney capsule limits the transplantation of sufficient islets in large animals and humans. The inguinal subcutaneous white adipose tissue (ISWAT), a new subcutaneous space, was found to be a potentially valuable site for islet transplantation. This site has better blood supply than other subcutaneous spaces. Moreover, the ISWAT accommodates a larger islet mass than the kidney capsule, and transplantation into it is simple. This manuscript describes the procedure of mouse islet isolation and transplantation in the ISWAT site of syngeneic diabetic mouse recipients. Using this protocol, murine pancreatic islets were isolated by standard collagenase digestion and a basement membrane matrix hydrogel was used for fixing the purified islets in the ISWAT site. The blood glucose levels of the recipient mice were monitored for more than 100 days. Islet grafts were retrieved at day 100 after transplantation for histological analysis. The protocol for islet transplantation in the ISWAT site described in this manuscript is simple and effective.

β-Cell and renal transplantation options for diabetes.

Despite major advances in structured education, insulin delivery and glucose monitoring, diabetes self-management remains an unremitting challenge. Insulin therapy is inextricably linked to risk of dangerous hypoglycaemia and sustained hyperglycaemia remains a leading cause of renal failure. This review sets out to demystify transplantation for diabetes multidisciplinary teams, facilitating consideration and incorporation within holistic overall person-centred management. Deceased and living donor kidney, whole pancreas and isolated islet transplant procedures, indications and potential benefits are described, in addition to outcomes within the integrated UK transplant programme.

BETA-2 score is an early predictor of graft decline and loss of insulin independence after pancreatic islet allotransplantation.

This study aimed to evaluate whether the BETA-2 score is a reliable early predictor of graft decline and loss of insulin independence after islet allotransplantation. Islet transplant procedures were stratified into 3 groups according to clinical outcome: long-term insulin independence without islet graft decline (group 1, N=9), initial insulin independence with subsequent islet graft decline and loss of insulin independence (group 2, N=13), and no insulin independence (group 3, N=13). BETA-2 was calculated on day 75 and multiple times afterwards for up to 145months posttransplantation. A BETA-2 score cut-off of 17.4 on day 75 posttransplantation was discerned between group 1 and groups 2 and 3 (area under the receiver operating characteristic 0.769, P=.005) with a sensitivity and negative predictive value of 100%. Additionally, BETA-2≥17.4 at any timepoint during follow-up reflected islet function required for long-term insulin independence. While BETA-2 did not decline below 17.4 for each of the 9 cases from group 1, the score decreased below 17.4 for all transplants from group 2 with subsequent loss of insulin independence. The reduction of BETA-2 below 17.4 predicted 9 (1.5-21)months in advance subsequent islet graft decline and loss of insulin independence (P=.03). This finding has important implications for posttransplant monitoring and patient care.

A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver

Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved. We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice. In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice. Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

Carnosine protects pancreatic beta cells and islets against oxidative stress damage.

Islet transplantation is a valid therapeutic option for type 1 diabetes treatment. However, in this procedure one of the major problems is the oxidative stress produced during pancreatic islet isolation. The aim of our study was to evaluate potential protective effects of L-carnosine and its isomer D-carnosine against oxidative stress. We evaluated the carnosine effect on cell growth, cell death, insulin production, and the main markers of oxidative stress in rat and murine stressed beta cell lines as well as in human pancreatic islets. Both isomers clearly inhibited hydrogen peroxide induced cytotoxicity, with a decrease in intracellular reactive oxygen and nitrogen species, prevented hydrogen peroxide induced apoptosis/necrosis, nitrite production, and reduced glucose-induced insulin secretion. In addition, NF-κB expression/translocation and nitrated protein induced in stressed cells was significantly reduced. Furthermore, both isomers improved survival and function, and decreased reactive oxygen and nitrogen species, and nitrite and nitrotyrosine production in human islets cultured for 1, 3, and 7 days. These results seem to indicate that both L and D-carnosine have a significant cytoprotective effect by reducing oxidative stress in beta cell lines and human islets, suggesting their potential use to improve islet survival during the islet transplantation procedure.

Introducing a New Experimental Islet Transplantation Model using Biomimetic Hydrogel and a Simple High Yield Islet Isolation Technique.

Background:Islet transplantation could be an ideal alternative treatment to insulin therapy for type 1 diabetes Mellitus (T1DM). This clinical and experimental field requires a model that covers problems such as requiring a large number of functional and viable islets, the optimal transplantation site, and the prevention of islet dispersion. Hence, the methods of choice for isolation of functional islets and transplantation are crucial.Methods:The present study has introduced an experimental model that overcomes some critical issues in islet transplantation, including in situ pancreas perfusion by digestive enzymes through common bile duct. In comparison with conventional methods, we inflated the pancreas in Petri dishes with only 1 ml collagenase type XI solution, which was followed by hand-picking isolation or Ficoll gradient separation to purify the islets. Then we used a hydrogel composite in which the islets were embedded and transplanted into the peritoneal cavity of the streptozotocin-induced diabetic C57BL/6 mice.Results:As compared to the yield of the classical methods, in our modified technique, the mean yield of isolation was about 130-200 viable islets/mouse pancreas. In vitro glucose-mediated insulin secretion assay indicated an appropriate response in isolated islets. In addition, data from in vivo experiments revealed that the allograft remarkably maintained blood glucose levels under 400 mg/dl and hydrogel composite prevents the passage of immune cells.Conclusion:In the model presented here, the rapid islet isolation technique and the application of biomimetic hydrogel wrapping of islets could facilitate islet transplantation procedures.

Cost and clinical outcome of islet transplantation in Norway 2010-2015.

Islet transplantation is a minimally invasive β-cell replacement strategy. Islet transplantation is a reimbursed treatment in Norway. Here, we summarize the cost and clinical outcome of 31 islet transplantations performed at Oslo University Hospital (OUS) from January 2010 to June 2015. Patients were retrospectively divided into three groups. Thirteen patients received either one or two islet transplantation alone (ITA), while five patients received islet transplantation after previous solid organ transplantation. For the group receiving 2 ITA, Kaplan-Meier estimates show an insulin independence of 20% more than 4years after their last transplantation. An estimated 70% maintain at least partial graft function, defined as fasting C-peptide >0.1nmolL-1 , and 47% maintain a HbA1c below 6.5% or 2 percent points lower than before ITA. For all groups combined, we estimate that 44% of the patients have a 50% reduction in insulin requirement 4years after the initial islet transplantation. The average cost for an islet transplantation procedure was 347297±60588NOK, or 35424±6182EUR, of which isolation expenses represent 34%. We hereby add to the common pool of growing experience with islet transplantation and also describe the cost of the treatment at our center.

Functional Improvement in Rats' Pancreatic Islets Using Magnesium Oxide Nanoparticles Through Antiapoptotic and Antioxidant Pathways.

According to undiscovered toxicity and safety of magnesium oxide nanoparticles (MgO NPs) in isolated pancreatic islet cells, this study was designed to examine the effects of its various concentrations on a time-course basis on the oxidative stress, viability, and function of isolated islets of rat's pancreas. Pancreatic islets were isolated and exposed to different MgO NP (<100nm) concentrations within three different time points. After that, oxidative stress biomarkers were investigated and the best exposure time was selected. Then, safety of MgO NPs was investigated by flow cytometry and fluorescent staining, and levels of insulin secretion and caspase activity were measured. The results illustrated a considerable decrease in oxidative stress markers such as reactive oxygen species (ROS) and lipid peroxidation (LPO) levels of pancreatic islets which were treated by MgO NPs for 24h. Also, in that time of exposure, cell apoptosis investigation by flow cytometry and insulin test showed that MgO NPs, in a concentration of 100μg/ml, decreased the rate of apoptotic cells via inhibiting caspase-9 activity and made a significant increase in the level of insulin secretion. Data of function and apoptosis biomarkers correlated with each other. It is concluded that the use of MgO NPs in concentration of as low as 100μg/ml can induce antiapoptotic, antioxidative, and antidiabetic effects in rat pancreatic islets, which support its possible benefit in islet transplantation procedures.

An Engineered Innate Repair Receptor Agonist, ARA 290, Protects Rat Islets from Cytokine-induced Apoptosis

The efficacy of pancreatic islet transplantation (PITx) is reduced due to severe inflammatory responses triggered by the islet isolation and transplantation procedures. ARA 290, an innate repair receptor agonist, has anti-apoptotic and anti-inflammatory properties and it has been shown to improve clinical symptoms caused by inflammation in sarcoidosis patients and type 2 diabetes. We here investigated whether ARA 290 treatment would improve the efficacy of PITx in a rat pancreatic islet transplantation model. For islets co-cultured with proinflammatory cytokines, addition of ARA290 showed better viability [percent of naive control in MTT assay: 54.6 ± 5.2 vs. 75.2 ± 6.4], insulin release [stimulation index in static glucose stimulated insulin secretion tests of 1.05 ± 0.63 vs. 2.61 ± 0.89], and reduced apoptosis [caspase3/7 activity182 ± 18 vs. 152 ± 18 luminescence/dsDNA(ng)] (islets with cytokines vs. islets with ARA290+cytokines, respectively, all p<0.05). In order to mimic the clinical situation, islets were isolated after prolonged cold ischemia (18 h). The addition of ARA 290 into preservation solution, however, did not improve islet yields or function. As a marginal syngeneic PITx, 220 syngeneic rat islets were transplanted under the kidney capsule of streptozotocin-induced diabetic rats and the recipients were treated with ARA 290 (120 μg/kg/day) for two weeks after PITx. In this model, no beneficial effect of ARA 290 treatment was observed. Our results indicated that ARA 290 protected rat pancreatic islets from cytokine-induced damage and apoptosis, but did not improve PITx perhaps due to poor penetration of ARA290 into transplant site or low IRR expression. ARA 290 could be useful as an agent to reduce islet injury caused by severe inflammation.

Sensing and Sensibility: Single-Islet-based Quality Control Assay of Cryopreserved Pancreatic Islets with Functionalized Hydrogel Microcapsules.

Despite decades of research and clinical studies of islet transplantations, finding simple yet reliable islet quality assays that correlate accurately with in vivo potency is still a major challenge, especially for real-time and single-islet-based quality assessment. Herein, proof-of-concept studies of a cryopreserved microcapsule-based quality control assays are presented for single islets. Individual rat pancreatic islets and fluorescent oxygen-sensitive dye (FOSD) are encapsulated in alginate hydrogel microcapsules via a microfluidic device. To test the susceptibility of the microcapsules and the FOSD to cryopreservation, the islet microcapsules containing FOSD are cryopreserved and the islet functionalities (adenosine triphosphate, static insulin release measurement, and oxygen consumption rate) are assessed after freezing and thawing steps. The cryopreserved islet capsules with FOSD remain functional after encapsulation and freezing/thawing procedures, validating a simple yet reliable individual-islet-based quality control method for the entire islet processing procedure prior to transplantation. This work also demonstrates that the functionality of cryopreserved islets can be improved by introducing trehalose into the routinely used cryoprotectant dimethyl sulfoxide. The functionalized alginate hydrogel microcapsules with embedded FOSD and optimized cryopreservation protocol presented in this work serve as a versatile islet quality assay and offer tremendous promise for tackling existing challenges in islet transplantation procedures.

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Humoral Immune Response following Seasonal Influenza Vaccine in Islet Transplant Recipients

Annual influenza vaccine is recommended for organ transplant recipients, but immunogenicity is known to be suboptimal. Islet transplant recipients receive immunosuppressive therapy, but there are no data on the immunogenicity of influenza vaccine in this population. In this prospective cohort study, adult islet transplant recipients at least 3 months posttransplant were enrolled. All patients received the 2010-2011 seasonal influenza vaccine. Serum was obtained pre- and postvaccination to determine humoral response to each of the three influenza strains included in the vaccine. Adverse effects of vaccine were also noted. A total of 61 islet transplant recipients were enrolled and completed the study protocol. The median time from last transplant was 1.9 years (range 0.26-11.4 years), and most patients had undergone multiple prior islet transplant procedures (90.2%). Overall immunogenicity of the vaccine was poor. Seroconversion rates to H1N1, H3N2, and B antigens were 34.4%, 29.5%, and 9.8%, respectively. In the subset not seroprotected at baseline, a protective antibody titer postvaccination was achieved in 58.6%, 41.9%, and 34.5% of patients, respectively. Patients within the first year of transplant were significantly less likely to seroconvert to at least one antigen (23.5% vs. 54.5%; p = 0.029). Alemtuzumab recipients trended toward lower seroconversion rates (25% vs. 51%; p = 0.11). No vaccine-related safety concerns were identified. Seasonal influenza vaccine had suboptimal immunogenicity in islet transplant recipients especially those who were less than 1 year posttransplant or had received alemtuzumab induction. Novel strategies for protection in this group of patients need further study.

Through a mechanism-based approach, nanoparticles of cerium and yttrium may improve the outcome of pancreatic islet isolation

Insulin dependent diabetes mellitus (IDDM) is a global disease with high economic and social burden. A potential cure for type 1 diabetes is pancreatic islet transplantation. Despite recent improvements, islets are faced with various types of stress related to the isolation and transplantation procedure. Oxidative stress plays an essential role in cell damage during islet isolation and transplantation procedures. In this article, we hypothesise that a combination of cerium and yttrium oxide nanoparticles, as a power antioxidant with free-radical-scavenging activity, because of enhancing the survival or viability of cells in vitro can improve islet transplantation. No doubt, administration of this improvement can be used in diabetes treatment.

Portal Vein Thrombosis Is a Potentially Preventable Complication in Clinical Islet Transplantation

Percutaneous transhepatic portal access avoids surgery but is rarely associated with bleeding or portal venous thrombosis (PVT). We herein report our large, single-center experience of percutaneous islet implantation and evaluate risk factors of PVT and graft function. Prospective data were collected on 268 intraportal islet transplants (122 subjects). A portal venous Doppler ultrasound was obtained on Days 1 and 7 posttransplant. Therapeutic heparinization, complete ablation of the portal catheter tract with Avitene paste and limiting packed cell volume (PCV) to <5 mL completely prevented any portal thrombosis in the most recent 101 islet transplant procedures over the past 5 years. In the previous cumulative experience, partial thrombosis did not affect islet function. Standard liver volume correlated negatively (r =-0.257, p < 0.001) and PCV correlated positively with portal pressure rise (r = 0.463, p < 0.001). Overall, partial portal thrombosis occurred after 10 procedures (overall incidence 3.7%, most recent 101 patient incidence 0%). There were no cases of complete thrombosis and no patient developed sequelae of portal hypertension. In conclusion, portal thrombosis is a preventable complication in clinical islet transplantation, provided therapeutic anticoagulation is maintained and PCV is limited to <5 mL.

Anthocyanins from Chinese Bayberry Extract Protect β Cells from Oxidative Stress-Mediated Injury via HO-1 Upregulation

Oxidative stress plays a pivotal role during the islet transplantation procedure, and antioxidant supplementation may protect grafts against oxidative injury. Chinese bayberry is one of six Myrica species native to China, and we demonstrated here that anthocyanins from Chinese bayberry extract (CBE) protect pancreatic β cells (INS-1) against hydrogen peroxide (H(2)O(2))-induced necrosis and apoptosis. Anthocyanins time- and dose-dependently upregulated heme oxygenase-1 (HO-1) gene expression in β cells and primary islets. HO-1 knockdown increased H(2)O(2)-induced cell death and attenuated the cytoprotective effect of anthocyanins. Anthocyanin treatment activated ERK1/2 and PI3K/Akt signaling, and ERK1/2 and PI3K inhibitors partially attenuated anthocyanin-mediated induction of HO-1. Additionally, β cells pretreated with anthocyanins displayed a decreased extent of apoptosis after transplantation. In summary, these results suggest that anthocyanins in CBE protect β cells from H(2)O(2)-induced cell injury via ERK1/2- and PI3K/Akt-mediated HO-1 upregulation.

Impact of pancreas transplantation on type 1 diabetes-related complications

Type 1 diabetes is a chronic disease that can impact patient survival and quality of life because of acute and chronic complications. Although intensive insulin scheme treatment has been shown to reduce the incidence of diabetes-related complications, only pancreas transplantation has been shown to be able to alter them and in some cases to revert them. In this review, an extensive view of the effect of pancreas transplantation on diabetes-related complication will be described. This review will focus on patients survival, diabetic nephropathy, neuropathy, cardiovascular event, comparing their incidence in type 1 diabetic patients treated with insulin and in type 1 diabetic patients receiving kidney, kidney-pancreas or pancreas alone graft. The review will focus mostly on the papers published in the last decade, with a particular attention to those on new aspects of graft function analysis like spectroscopy. Moreover, a comparison with islet transplantation procedure will be performed. This review will give an update on the potential of pancreas transplantation, give a guide for clinical practice and help to consider pancreas transplantation as an alternative to insulin treatment for selected patients.

Current Advances and Travails in Islet Transplantation

The successful demonstration that insulin-producing β-cells can be isolated (in the form of cell clusters called islets containing β and other endocrine and nonendocrine cells) from a recently deceased donor's pancreas, then transplanted into subjects with type 1 diabetes, and thereby restore, at least temporarily, insulin-independent normoglycemia has firmly established the important “proof of concept.” Even so, worldwide efforts to advance the therapy for widespread applicability have served to focus attention on the hurdles yet to clear. This review will briefly describe the present state of the art and succinctly define the research problems being attacked along with some recent advances that demonstrate significant progress. Since Paul Lacy's early rodent experiments in the 1960s established that pancreatic islets could be isolated from one animal and transplanted into a diabetic recipient to restore normoglycemia (1), investigators have pursued efforts to develop the therapy for clinical use. After years of development in various animal models and efforts to improve human islet isolation techniques (see [2–4] for reviews with a historical perspective), the first patient achieving short-term insulin independence was reported by the group at Washington University in St. Louis. That advance was based on new islet isolation technology utilizing islets pooled from several donors, intensive insulin treatment in the peritransplant period, and induction immunosuppression with antithymocyte globulin (ATG) to avoid glucocorticoid therapy (5). The development of new immunosuppressive drugs that allowed patients to remain off glucocorticoid therapy while awaiting subsequent islet infusions (because most recipients require islets from two or more donors) enabled the group in Edmonton to optimize the clinical islet transplantation procedure (6). The approach allowed the group to conclude that about 12,000 islet equivalents per recipient body weight (in kilograms) was required to restore insulin-independent normoglycemia (6) and sparked intense international interest and effort. Current …