Transplantation of macroencapsulated human islets within the bioartificial pancreas βAir to patients with type 1 diabetes mellitus

Macroencapsulation devices provide the dual possibility of immunoprotecting transplanted cells while also being retrievable, the latter bearing importance for safety in future trials with stem cell–derived cells. However, macroencapsulation entails a problem with oxygen supply to the encapsulated cells. The βAir device solves this with an incorporated refillable oxygen tank. This phase 1 study evaluated the safety and efficacy of implanting the βAir device containing allogeneic human pancreatic islets into patients with type 1 diabetes. Four patients were transplanted with 1‐2 βAir devices, each containing 155 000‐180 000 islet equivalents (ie, 1800‐4600 islet equivalents per kg body weight), and monitored for 3‐6 months, followed by the recovery of devices. Implantation of the βAir device was safe and successfully prevented immunization and rejection of the transplanted tissue. However, although beta cells survived in the device, only minute levels of circulating C‐peptide were observed with no impact on metabolic control. Fibrotic tissue with immune cells was formed in capsule surroundings. Recovered devices displayed a blunted glucose‐stimulated insulin response, and amyloid formation in the endocrine tissue. We conclude that the βAir device is safe and can support survival of allogeneic islets for several months, although the function of the transplanted cells was limited (Clinicaltrials.gov: NCT02064309).     

Posttransplant oxygen inhalation improves the outcome of subcutaneous islet transplantation: A promising clinical alternative to the conventional intrahepatic site

Subcutaneous tissue is a promising site for islet transplantation, due to its large area and accessibility, which allows minimally invasive procedures for transplantation, graft monitoring, and removal of malignancies as needed. However, relative to the conventional intrahepatic transplantation site, the subcutaneous site requires a large number of islets to achieve engraftment success and diabetes reversal, due to hypoxia and low vascularity. We report that the efficiency of subcutaneous islet transplantation in a Lewis rat model is significantly improved by treating recipients with inhaled 50% oxygen, in conjunction with prevascularization of the graft bed by agarose–basic fibroblast growth factor. Administration of 50% oxygen increased oxygen tension in the subcutaneous site to 140 mm Hg, compared to 45 mm Hg under ambient air. In vitro, islets cultured under 140 mm Hg oxygen showed reduced central necrosis and increased insulin release, compared to those maintained in 45 mm Hg oxygen. Six hundred syngeneic islets subcutaneously transplanted into the prevascularized graft bed reversed diabetes when combined with postoperative 50% oxygen inhalation for 3 days, a number comparable to that required for intrahepatic transplantation; in the absence of oxygen treatment, diabetes was not reversed. Thus, we show oxygen inhalation to be a simple and promising approach to successfully establishing subcutaneous islet transplantation.     

Noninvasive diagnosis of recurrent autoimmune type 1 diabetes after islet cell transplantation

Islet cell transplantation is curative therapy for patients with complicated autoimmune type 1 diabetes (T1D). We report the diagnostic potential of circulating transplant islet–specific exosomes to noninvasively distinguish pancreatic β cell injury secondary to recurrent autoimmunity vs immunologic rejection. A T1D patient with hypoglycemic unawareness underwent islet transplantation and maintained normoglycemia until posttransplant day 1098 before requiring exogenous insulin. Plasma analysis showed decreased donor islet exosome quantities on day 1001, before hyperglycemia onset. This drop in islet exosome quantity signified islet injury, but did not distinguish injury type. However, analysis of purified transplant islet exosome cargoes showed decrease in insulin‐containing exosomes, but not glucagon‐containing exosomes, indicating selective destruction of transplanted β cells secondary to recurrent T1D autoimmunity. Furthermore, donor islet exosome cargo analysis showed time‐specific increase in islet autoantigen, glutamic acid decarboxylase 65 (GAD65), implicated in T1D autoimmunity. Time‐matched analysis of plasma transplant islet exosomes in 3 control subjects undergoing islet cell transplantation failed to show changes in islet exosome quantities or intraexosomal cargo expression of insulin, glucagon, and GAD65. This is the first report of noninvasive diagnosis of recurrent autoimmunity after islet cell transplantation, suggesting that transplant tissue exosome platform may serve as a biomarker in islet transplant diagnostics.     

Shielding islets with human amniotic epithelial cells enhances islet engraftment and revascularization in a murine diabetes model

Hypoxia is a major cause of considerable islet loss during the early posttransplant period. Here, we investigate whether shielding islets with human amniotic epithelial cells (hAECs), which possess anti‐inflammatory and regenerative properties, improves islet engraftment and survival. Shielded islets were generated on agarose microwells by mixing rat islets (RIs) or human islets (HI) and hAECs (100 hAECs/IEQ). Islet secretory function and viability were assessed after culture in hypoxia (1% O₂) or normoxia (21% O₂) in vitro. In vivo function was evaluated after transplant under the kidney capsule of diabetic immunodeficient mice. Graft morphology and vascularization were evaluated by immunohistochemistry. Both shielded RIs and HIs show higher viability and increased glucose‐stimulated insulin secretion after exposure to hypoxia in vitro compared with control islets. Transplant of shielded islets results in considerably earlier normoglycemia and vascularization, an enhanced glucose tolerance, and a higher β cell mass. Our results show that hAECs have a clear cytoprotective effect against hypoxic damages in vitro. This strategy improves β cell mass engraftment and islet revascularization, leading to an improved capacity of islets to reverse hyperglycemia, and could be rapidly applicable in the clinical situation seeing that the modification to HIs are minor.     

Loss of end‐differentiated β‐cell phenotype following pancreatic islet transplantation

Replacement of pancreatic β‐cells through deceased donor islet transplantation is a proven therapy for preventing recurrent life‐threatening hypoglycemia in type 1 diabetes. Although near‐normal glucose levels and insulin independence can be maintained for many years following successful islet transplantation, restoration of normal functional β‐cell mass has remained elusive. It has recently been proposed that dedifferentiation/plasticity towards other endocrine phenotypes may play an important role in stress‐induced β‐cell dysfunction in type 2 diabetes. Here we report loss of end‐differentiated β‐cell phenotype in 2 intraportal islet allotransplant recipients. Despite excellent graft function and sustained insulin independence, all examined insulin‐positive cells had lost expression of the end‐differentiation marker, urocortin‐3, or appeared to co‐express the α‐cell marker, glucagon. In contrast, no insulin⁺/urocortin‐3^? cells were seen in nondiabetic deceased donor control pancreatic islets. Loss of end‐differentiated phenotype may facilitate β‐cell survival during the stresses associated with islet isolation and culture, in addition to sustained hypoxia following engraftment. As further refinements in islet isolation and culture are made in parallel with exploration of alternative β‐cell sources, graft sites, and ultimately fully vascularized bioengineered insulin‐secreting microtissues, differentiation status immunostaining provides a novel tool to assess whether fully mature β‐cell phenotype has been maintained.     

Clinical use of donation after circulatory death pancreas for islet transplantation

Due to a shortage of donation after brain death (DBD) organs, donation after circulatory death (DCD) is increasingly performed. In the field of islet transplantation, there is uncertainty regarding the suitability of DCD pancreas in terms of islet yield and function after islet isolation. The aim of this study was to investigate the potential use of DCD pancreas for islet transplantation. Islet isolation procedures from 126 category 3 DCD and 258 DBD pancreas were performed in a 9-year period. Islet yield after isolation was significantly lower for DCD compared to DBD pancreas (395 515 islet equivalents [IEQ] and 480 017 IEQ, respectively; p = .003). The decrease in IEQ during 2 days of culture was not different between the two groups. Warm ischemia time was not related to DCD islet yield. In vitro insulin secretion after a glucose challenge was similar between DCD and DBD islets. After islet transplantation, DCD islet graft recipients had similar graft function (AUC C-peptide) during mixed meal tolerance tests and Igls score compared to DBD graft recipients. In conclusion, DCD islets can be considered for clinical islet transplantation.     

Rapid Restoration of Vascularity and Oxygenation in Mouse and Human Islets Transplanted to Omentum May Contribute to Their Superior Function Compared to Intraportally Transplanted Islets

Transplantation of islets into the liver confers several site‐specific challenges, including a delayed vascularization and prevailing hypoxia. The greater omentum has in several experimental studies been suggested as an alternative implantation site for clinical use, but there has been no direct functional comparison to the liver. In this experimental study in mice, we characterized the engraftment of mouse and human islets in the omentum and compared engraftment and functional outcome with those in the intraportal site. The vascularization and innervation of the islets transplanted into the omentum were restored within the first month by paralleled ingrowth of capillaries and nerves. The hypoxic conditions in the islets early posttransplantation were transient and restricted to the first days. Newly formed blood vessels were fully functional, and the blood perfusion and oxygenation of the islets became similar to that of endogenous islets. Furthermore, islet grafts in the omentum showed at 1 month posttransplantation functional superiority to intraportally transplanted grafts. We conclude that in contrast to the liver the omentum provides excellent engraftment conditions for transplanted islets. Future studies in humans will be of great interest to investigate the capability of this site to also harbor larger grafts without interfering with islet functionality.     

Ten-year outcomes of islet transplantation in patients with type 1 diabetes: Data from the Swiss-French GRAGIL network

To describe the 10-year outcomes of islet transplantation within the Swiss-French GRAGIL Network, in patients with type 1 diabetes experiencing high glucose variability associated with severe hypoglycemia and/or with functional kidney graft. We conducted a retrospective analysis of all subjects transplanted in the GRAGIL-1c and GARGIL-2 islet transplantation trials and analyzed components of metabolic control, graft function and safety outcomes over the 10-year period of follow-up. Forty-four patients were included between September 2003 and April 2010. Thirty-one patients completed a 10-year follow-up. Ten years after islet transplantation, median HbA1c was 7.2% (6.2–8.0) (55 mmol/mol [44–64]) versus 8.0% (7.1–9.1) (64 mmol/mol [54–76]) before transplantation (p < .001). Seventeen of 23 (73.9%) recipients were free of severe hypoglycemia, 1/21 patients (4.8%) was insulin-independent and median C-peptide was 0.6 ng/ml (0.2–1.2). Insulin requirements (UI/kg/day) were 0.3 (0.1–0.5) versus 0.5 (0.4–0.6) before transplantation (p < .001). Median (IQR) β-score was 1 (0–4) (p < .05 when comparing with pre-transplantation values) and 51.9% recipients had a functional islet graft at 10 years. With a 10-year follow-up in a multicentric network, islet transplantation provided sustained improvement of glycemic control and was efficient to prevent severe hypoglycemia in almost 75% of the recipients.     

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

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

Alpha‐1‐antitrypsin in cell and organ transplantation

Limited availability of donor organs and risk of ischemia‐reperfusion injury (IRI) seriously restrict organ transplantation. Therapeutics that can prevent or reduce IRI could potentially increase the number of transplants by increasing use of borderline organs and decreasing discards. Alpha‐1 antitrypsin (AAT) is an acute phase reactant and serine protease inhibitor that limits inflammatory tissue damage. Purified plasma–derived AAT has been well tolerated in more than 30 years of use to prevent emphysema in AAT‐deficient individuals. Accumulating evidence suggests that AAT has additional anti‐inflammatory and tissue‐protective effects including improving mitochondrial membrane stability, inhibiting apoptosis, inhibiting nuclear factor kappa B activation, modulating pro‐ vs anti‐inflammatory cytokine balance, and promoting immunologic tolerance. Cell culture and animal studies have shown that AAT limits tissue injury and promotes cell and tissue survival. AAT can promote tolerance in animal models by downregulating early inflammation and favoring induction and stabilization of regulatory T cells. The diverse intracellular and immune‐modulatory effects of AAT and its well‐established tolerability in patients suggest that it might be useful in transplantation. Clinical trials, planned and/or in progress, should help determine whether the promise of the animal and cellular studies will be fulfilled by improving outcomes in human organ transplantation.     

Islet damage during isolation as assessed by miRNAs and the correlation of miRNA levels with posttransplantation outcome in islet autotransplantation

High‐quality pancreatic islets are essential for better posttransplantation endocrine function in total pancreatectomy with islet autotransplantation (TPIAT), yet stress during the isolation process affects quality and yield. We analyzed islet‐enriched microRNAs (miRNAs) ‐375 and ‐200c released during isolation to assess damage and correlated the data with posttransplantation endocrine function. The absolute concentration of miR‐375, miR‐200c, and C‐peptide was measured in various islet isolation steps, including digestion, dilution, recombination, purification, and bagging, in 12 cases of TPIAT. Posttransplantation glycemic control was monitored through C‐peptide, hemoglobin A_1c, insulin requirement, and SUITO index. The amount of miR‐375 released was significantly higher during enzymatic digestion followed by the islet bagging (P < .001). Mir‐200c mirrored these changes, albeit at lower concentrations. In contrast, the C‐peptide amount was significantly higher in the purification and bagging steps (P < .001). Lower amounts of miR‐375 were associated with a lower 6‐month insulin requirement (P = .01) and lower hemoglobin A_1c (P = .04). Measurement of the absolute quantity of miRNA‐375 and ‐200c released during islet isolation is a useful tool to assess islet damage. The quantity of released miRNA is indicative of posttransplantation endocrine function in TPIAT patients.     

Novel cell‐permeable p38‐MAPK inhibitor efficiently prevents porcine islet apoptosis and improves islet graft function

During islet transplantation, mitogen‐activated protein kinase (MAPK) p38 is preferentially activated in response to the isolation of islets and the associated inflammation. Although therapeutic effects of p38 inhibitors are expected, the clinical application of small‐molecule inhibitors of p38 is not recommended because of their serious adverse effects on the liver and central nervous system. Here we designed peptides to inhibit p38, which were derived from the sites on p38 that mediate binding to proteins such as MAPK kinases. Peptide 11R‐p38I₁₁₀ significantly inhibited the activation of p38. To evaluate the effects of 11R‐p38I₁₁₀, porcine islets were incubated with 10 µmol/L 11R‐p38I₁₁₀ or a mutant form designated 11R‐mp38I₁₁₀. After islet transplantation, blood glucose levels reached the normoglycemic range in 58.3% and 0% of diabetic mice treated with 11R‐p38I₁₁₀ or 11R‐mp38I₁₁₀, respectively. These data suggest that 11R‐p38I₁₁₀ inhibited islet apoptosis and improved islet function. Peptide p38I₁₁₀ is a noncompetitive inhibitor of ATP and targets a unique docking site. Therefore, 11R‐p38I₁₁₀ specifically inhibits p38 activation, which may avoid the adverse effects that have discouraged the clinical use of small‐molecule inhibitors of p38. Moreover, our methodology to design “peptide inhibitors” could be used to design other inhibitors derived from the binding sites of proteins.     

Autoreactive T cell profiles are altered following allogeneic islet transplantation with alemtuzumab induction and re-emerging phenotype is associated with graft function

Islet transplantation is an effective therapy for life-threatening hypoglycemia, but graft function gradually declines over time in many recipients. We characterized islet-specific T cells in recipients within an islet transplant program favoring alemtuzumab (ATZ) lymphodepleting induction and examined associations with graft function. Fifty-eight recipients were studied: 23 pretransplant and 40 posttransplant (including 5 with pretransplant phenotyping). The proportion with islet-specific T cell responses was not significantly different over time (pre-Tx: 59%; 1–6 m posttransplant: 38%; 7–12 m: 44%; 13–24 m: 47%; and >24 m: 45%). However, phenotype shifted significantly, with IFN-γ–dominated response in the pretransplant group replaced by IL-10–dominated response in the 1–6 m posttransplant group, reverting to predominantly IFN-γ–oriented response in the >24 m group. Clustering analysis of posttransplant responses revealed two main agglomerations, characterized by IFN-γ and IL-10 phenotypes, respectively. IL-10–oriented posttransplant response was associated with relatively low graft function. Recipients within the IL-10⁺ cluster had a significant decline in C-peptide levels in the period preceding the IL-10 response, but stable graft function following the response. In contrast, an IFN-γ response was associated with subsequently decreased C-peptide. Islet transplantation favoring ATZ induction is associated with an initial altered islet-specific T cell phenotype but reversion toward pretransplant profiles over time. Posttransplant autoreactive T cell phenotype may be a predictor of subsequent graft function.     

Assessment of plasma microvesicles to monitor pancreatic islet graft dysfunction: Beta cell‐ and leukocyte‐derived microvesicles as specific features in a pilot longitudinal study

Markers of early pancreatic islet graft dysfunction and its causes are lacking. We monitored 19 type 1 diabetes islet‐transplanted patients for up to 36 months following last islet injection. Patients were categorized as Partial (PS) or complete (S) Success, or Graft Failure (F), using the β‐score as an indicator of graft function. F was the subset reference of maximum worsened graft outcome. To identify the immune, pancreatic, and liver contribution to the graft dysfunction, the cell origin and concentration of circulating microvesicles (MVs) were assessed, including MVs from insulin‐secreting β‐cells typified by polysialic acid of neural cell adhesion molecule (PSA‐NCAM), and data were compared with values of the β‐score. Similar ranges of PSA‐NCAM⁺‐MVs were found in healthy volunteers and S patients, indicating minimal cell damage. In PS, a 2‐fold elevation in PSA‐NCAM⁺‐MVs preceded each β‐score drop along with a concomitant rise in insulin needs, suggesting β‐cell damage or altered function. Significant elevation of liver asialoglycoprotein receptor (ASGPR)⁺‐MVs, endothelial CD105⁺‐MVs, neutrophil CD66b⁺‐MVs, monocyte CD 14⁺‐MVs, and T4 lymphocyte CD4⁺‐MVs occurred before each β‐score drop, CD8⁺‐MVs increased only in F, and B lymphocyte CD19⁺‐MVs remained undetectable. In conclusion, PSA‐NCAM⁺‐MVs are noninvasive early markers of transplant dysfunction, while ASGPR⁺‐MVs signal host tissue remodeling. Leukocyte MVs could identify the cause of graft dysfunction.     

Identification and Characterization of microRNAs Associated With Human β‐Cell Loss in a Mouse Model

Currently there is no effective approach for monitoring early β‐cell loss during islet graft rejection following human islet transplantation (HIT). Due to ethical and technical constraints, it is difficult to directly study biomarkers of islet destruction in humans. Here, we established a humanized mouse model with induced human β‐cell death using adoptive lymphocyte transfer (ALT). Human islet grafts of ALT‐treated mice had perigraft lymphocyte infiltration, fewer insulin⁺ β cells, and increased β‐cell apoptosis. Islet‐specific miR‐375 was used to validate our model, and expression of miR‐375 was significantly decreased in the grafts and increased in the circulation of ALT‐treated mice before hyperglycemia. A NanoString expression assay was further used to profile 800 human miRNAs in the human islet grafts, and the results were validated using quantitative real‐time polymerase chain reaction. We found that miR‐4454 and miR‐199a‐5p were decreased in the human islet grafts following ALT and increased in the circulation prior to hyperglycemia. These data demonstrate that our in vivo model of induced human β‐cell destruction is a robust method for identifying and characterizing circulating biomarkers, and suggest that miR‐4454 and miR‐199a‐5p can serve as novel biomarkers associated with early human β‐cell loss following HIT.     

Single center results of simultaneous pancreas-kidney transplantation in patients with type 2 diabetes

Studies have found similar outcomes of Simultaneous Pancreas-Kidney transplantation (SPKT) in patients with Type 2 (T2D) and Type 1 diabetes (T1D). However, there are scarce data evaluating the association of recipient factors such as age, BMI, or pretransplant insulin requirements with outcomes, thus the criteria for the optimal recipient selection remains unclear. In this study, 284 T1D and 39 T2D patients, who underwent SPKT between 2006 and 2017 with 1 year of follow-up at minimum, were assessed for potential relationship of pretransplant BMI and insulin requirements with posttransplant diabetes and pancreatic graft failure. Kaplan-Meier analysis showed similar rates of freedom from posttransplant diabetes (94.7% T2D vs. 92.3% T1D at 1 yr, and 88.1% T2D vs. 81.1% T1D at 5 yrs) and graft survival (89.7% T2D vs. 90.4% T1D at 1 yr, and 89.7% T2D vs. 81.2% T1D at 5 yrs). There was no significant association between BMI or pretransplant insulin requirements with posttransplant diabetes occurrence in either T1D (p = .10, .43, respectively) or T2D (p = .12, .63) patients in the cohort; or with graft failure (T1D: p = .40, .09; T2D: p = .71, .28). These observations suggest a less restricted approach to selective use of SPKT in patients with T2D.