An engineered macroencapsulation membrane releasing FTY720 to precondition pancreatic islet transplantation.

Macroencapsulation is a powerful approach to increase the efficiency of extrahepatic pancreatic islet transplant. FTY720, a small molecule that activates signaling through sphingosine-1-phosphate receptors, is immunomodulatory and pro-angiogenic upon sustained delivery from biomaterials. While FTY720 (fingolimod, Gilenya) has been explored for organ transplantation, in the present work the effect of locally released FTY720 from novel nanofiber-based macroencapsulation membranes is explored for islet transplantation. We screened islet viability during culture with FTY720 and various biodegradable polymers. Islet viability is significantly reduced by the addition of high doses (≥500 ng/mL) of soluble FTY720. Among the polymers screened, islets have the highest viability when cultured with poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Therefore, PHBV was blended with polycaprolactone (PCL) for mechanical stability and electrospun into nanofibers. Islets had no detectable function ex vivo following 5 days or 12 h of subcutaneous implantation within our engineered device. Subsequently, we explored a preconditioning scheme in which islets are transplanted 2 weeks after FTY720-loaded nanofibers are implanted. This allows FTY720 to orchestrate a local regenerative milieu while preventing premature transplantation into avascular sites that contain high concentrations of FTY720. These results provide a foundation and motivation for further investigation into the use of FTY720 in preconditioning sites for efficacious islet transplantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 555-568, 2018.

Deformability-based microfluidic separation of pancreatic islets from exocrine acinar tissue for transplant applications.

The long-term management of type-1 diabetes (T1D) is currently achieved through lifelong exogenous insulin injections. Although there is no cure for T1D, transplantation of pancreatic islets of Langerhans has the potential to restore normal endocrine function versus the morbidity of hypoglycemic unawareness that is commonly associated with sudden death among fragile diabetics. However, since endocrine islet tissues form a small proportion of the pancreas, sufficient islet numbers can be reached only by combining islets from multiple organ donors and the transplant plug contains significantly high levels of exocrine acinar tissue, thereby exacerbating immune responses. Hence, lifelong administration of immunosuppressants is required after transplantation, which can stress islet cells. The density gradient method that is currently used to separate islets from acinar tissue causes islets to be sparsely distributed over the centrifuged bins, so that the transplant sample obtained by combining multiple bins also contains significant acinar tissue levels. We show that in comparison to the significant size and density overlaps between the islet and acinar tissue populations post-organ digestion, their deformability overlaps are minimal. This feature is utilized to design a microfluidic separation strategy, wherein tangential flows enable selective deformation of acinar populations towards the bifurcating waste stream and sequential switching of hydrodynamic resistance enables the collection of rigid islets. Using 25 bifurcating daughter channels, a throughput of ∼300 islets per hour per device is obtained for enabling islet enrichment from relatively dilute starting levels to purity levels that meet the transplant criteria, as well as to further enhance islet purity from samples following density gradient enrichment. Based on confirmation of viability and functionality of the microfluidic-isolated islets using insulin secretion analysis and an angiogenesis assay, we envision utilizing this strategy to generate small-volume transplant plugs with high islet purity and significantly reduced acinar levels for minimizing immune responses after transplantation.

STEAP4 expression in human islets is associated with differences in body mass index, sex, HbA1c, and inflammation.

OBJECTIVE: STEAP4 (six-transmembrane epithelial antigen of the prostate 4) is a metalloreductase that has been shown previously to protect cells from inflammatory damage. Genetic variants in STEAP4 have been associated with numerous metabolic disorders related to obesity, including putative defects in the acute insulin response to glucose in type 2 diabetes. PURPOSE: We examined whether obesity and/or type 2 diabetes altered STEAP4 expression in human pancreatic islets. METHODS: Human islets were isolated from deceased donors at two medical centers and processed for quantitative polymerase chain reaction. Organ donors were selected by status as non-diabetic or having type 2 diabetes. Site 1 (Edmonton): N = 13 type 2 diabetes donors (7M, 6F), N = 20 non-diabetic donors (7M, 13F). Site 2 (Virginia): N = 6 type 2 diabetes donors (6F), N = 6 non-diabetic donors (3M, 3F). RESULTS: STEAP4 showed reduced islet expression with increasing body mass index among all donors (P < 0.10) and non-diabetic donors (P < 0.05) from Site 1; STEAP4 showed reduced islet expression among type 2 diabetes donors with increasing hemoglobin A1c. Islet STEAP4 expression was also marginally higher in female donors (P < 0.10). Among type 2 diabetes donors from Site 2, islet insulin expression was reduced, STEAP4 expression was increased, and white blood cell counts were increased compared to non-diabetic donors. Islets from non-diabetic donors that were exposed overnight to 5 ng/ml IL-1ß displayed increased STEAP4 expression, consistent with STEAP4 upregulation by inflammatory signaling. CONCLUSIONS: These findings suggest that increased STEAP4 mRNA expression is associated with inflammatory stimuli, whereas lower STEAP4 expression is associated with obesity in human islets. Given its putative protective role, downregulation of STEAP4 by chronic obesity suggests a mechanism for reduced islet protection against cellular damage.

The impact of bacterial colonization on graft success after total pancreatectomy with autologous islet transplantation: considerations for early definitive surgical intervention.

OBJECTIVE: The purpose of this study was to determine whether bacterial contamination of islets affects graft success after total pancreatectomy with islet autotransplantation (TPIAT). BACKGROUND: Factors associated with insulin independence after TPIAT are inconclusive. Although bacterial contamination does not preclude transplantation, the impact of bacterial contamination on graft success is unknown. METHODS: Patients who received TPIAT at the University of Virginia between January 2007 and January 2016 were reviewed. Patient charts were reviewed for bacterial contamination and patients were prospectively contacted to assess rates of insulin independence. RESULTS: There was no significant difference in demographic or perioperative data between patients who achieved insulin independence and those who did not. However, six of 27 patients analyzed (22.2%) grew bacterial contaminants from culture of the final islet preparations. These patients had significantly lower islet yield and C-peptide at most recent follow-up (P<.05), and none of these patients achieved insulin independence. CONCLUSIONS: Islet transplant solutions are often culture positive, likely secondary to preprocurement pancreatic manipulation and introduction of enteric flora. Although autotransplantation of culture-positive islets is safe, it is associated with higher rates of graft failure and poor islet yield. Consideration should be given to identify patients who may develop refractory chronic pancreatitis and offer early operative management to prevent bacterial colonization.

Human ß cell transcriptome analysis uncovers lncRNAs that are tissue-specific, dynamically regulated, and abnormally expressed in type 2 diabetes.

A significant portion of the genome is transcribed as long noncoding RNAs (lncRNAs), several of which are known to control gene expression. The repertoire and regulation of lncRNAs in disease-relevant tissues, however, has not been systematically explored. We report a comprehensive strand-specific transcriptome map of human pancreatic islets and ß cells, and uncover >1100 intergenic and antisense islet-cell lncRNA genes. We find islet lncRNAs that are dynamically regulated and show that they are an integral component of the ß cell differentiation and maturation program. We sequenced the mouse islet transcriptome and identify lncRNA orthologs that are regulated like their human counterparts. Depletion of HI-LNC25, a ß cell-specific lncRNA, downregulated GLIS3 mRNA, thus exemplifying a gene regulatory function of islet lncRNAs. Finally, selected islet lncRNAs were dysregulated in type 2 diabetes or mapped to genetic loci underlying diabetes susceptibility. These findings reveal a new class of islet-cell genes relevant to ß cell programming and diabetes pathophysiology.

Adenosine A(2A) agonist administration improves islet transplant outcome: Evidence for the role of innate immunity in islet graft rejection.

Activation of adenosine A(2A) receptors inhibits inflammation in ischemia/reperfusion injury, and protects against cell damage at the injury site. Following transplantation 50% of islets die due to inflammation and apoptosis. This study investigated the effects of adenosine A(2A) receptor agonists (ATL146e and ATL313) on glucose-stimulated insulin secretion (GSIS) in vitro and transplanted murine syngeneic islet function in vivo. Compared to vehicle controls, ATL146e (100 nM) decreased insulin stimulation index [SI, (insulin)(high glucose)/(insulin)(low glucose)] (2.36 +/- 0.22 vs. 3.75 +/- 0.45; n = 9; p < 0.05). Coculture of islets with syngeneic leukocytes reduced SI (1.41 +/- 0.17; p < 0.05), and this was restored by ATL treatment (2.57 +/- 0.18; NS). Addition of a selective A(2A)AR antagonist abrogated ATL's protective effect, reducing SI (1.11 +/- 0.42). ATL treatment of A(2A)AR(+/+) islet/A(2A)AR(-/-) leukocyte cocultures failed to protect islet function (SI), implicating leukocytes as likely targets of A(2A)AR agonists. Diabetic recipient C57BL/6 mice (streptozotocin; 250 mg/kg, IP) received islet transplants to either the renal subcapsular or hepatic-intraportal site. Recipient mice receiving ATL therapy (ATL 146e or ATL313, 60 ng/kg/min, IP) achieved normoglycemia more rapidly than untreated recipients. Histological examination of grafts suggested reduced cellular necrosis, fibrosis, and lymphocyte infiltration in agonist-treated animals. Administration of adenosine A(2A) receptor agonists (ATL146e or ATL313) improves in vitro GSIS by an effect on leukocytes, and improves survival and functional engraftment of transplanted islets by inhibiting inflammatory islet damage in the peritransplant period, suggesting a potentially significant new strategy for reducing inflammatory islet loss in clinical transplantation.