R. Damaris Molano

A novel method for the assessment of cellular composition and beta-cell viability in human islet preparations

Current methodologies to evaluate islet cell viability are largely based on tests that assess the exclusion of DNA‐binding dyes. While these tests identify cells that have lost selective membrane permeability, they do not allow us to recognize apoptotic cells, which do not yet stain with DNA‐binding dyes. Furthermore, current methods of analysis do not discriminate between cell subsets in the preparation and, in particular, they do not allow for selectively defining β‐cell viability.

Noninvasive in vivo imaging of pancreatic islet cell biology

Advanced imaging techniques have become a valuable tool in the study of complex biological processes at the cellular level in biomedical research. Here, we introduce a new technical platform for noninvasive in vivo fluorescence imaging of pancreatic islets using the anterior chamber of the eye as a natural body window. Islets transplanted into the mouse eye engrafted on the iris, became vascularized, retained cellular composition, responded to stimulation and reverted diabetes. Laser-scanning microscopy allowed repetitive in vivo imaging of islet vascularization, beta cell function and death at cellular resolution. Our results thus establish the basis for noninvasive in vivo investigations of complex cellular processes, like beta cell stimulus-response coupling, which can be performed longitudinally under both physiological and pathological conditions.

Recurrence of type 1 diabetes after simultaneous pancreas-kidney transplantation, despite immunosuppression, is associated with autoantibodies and pathogenic autoreactive CD4 T-cells

OBJECTIVE To investigate if recurrent autoimmunity explained hyperglycemia and C-peptide loss in three immunosuppressed simultaneous pancreas-kidney (SPK) transplant recipients. RESEARCH DESIGN AND METHODS We monitored autoantibodies and autoreactive T-cells (using tetramers) and performed biopsy. The function of autoreactive T-cells was studied with in vitro and in vivo assays. RESULTS Autoantibodies were present pretransplant and persisted on follow-up in one patient. They appeared years after transplantation but before the development of hyperglycemia in the remaining patients. Pancreas transplant biopsies were taken within ∼1 year from hyperglycemia recurrence and revealed β-cell loss and insulitis. We studied autoreactive T-cells from the time of biopsy and repeatedly demonstrated their presence on further follow-up, together with autoantibodies. Treatment with T-cell–directed therapies (thymoglobulin and daclizumab, all patients), alone or with the addition of B-cell–directed therapy (rituximab, two patients), nonspecifically depleted T-cells and was associated with C-peptide secretion for >1 year. Autoreactive T-cells with the same autoantigen specificity and conserved T-cell receptor later reappeared with further C-peptide loss over the next 2 years. Purified autoreactive CD4 T-cells from two patients were cotransplanted with HLA-mismatched human islets into immunodeficient mice. Grafts showed β-cell loss in mice receiving autoreactive T-cells but not control T-cells. CONCLUSIONS We demonstrate the cardinal features of recurrent autoimmunity in three such patients, including the reappearance of CD4 T-cells capable of mediating β-cell destruction. Markers of autoimmunity can help diagnose this underappreciated cause of graft loss. Immune monitoring during therapy showed that autoimmunity was not resolved by the immunosuppressive agents used.

Reversal of Diabetes by Pancreatic Islet Transplantation into a Subcutaneous, Neovascularized Device

Background. Transplantation of pancreatic islets for the treatment of type 1 diabetes allows for physiologic glycemic control and insulin-independence when sufficient islets are implanted via the portal vein into the liver. Intrahepatic islet implantation requires specific infrastructure and expertise, and risks inherent to the procedure include bleeding, thrombosis, and elevation of portal pressure. Additionally, the relatively higher drug metabolite concentrations in the liver may contribute to the delayed loss of graft function of recent clinical trials. Identification of alternative implantation sites using biocompatible devices may be of assistance improving graft outcome. A desirable bioartificial pancreas should be easy to implant, biopsy, and retrieve, while allowing for sustained graft function. The subcutaneous (SC) site may require a minimally invasive procedure performed under local anesthesia, but its use has been hampered so far by lack of early vascularization, induction of local inflammation, and mechanical stress on the graft. Methods. Chemically diabetic rats received syngeneic islets into the liver or SC into a novel biocompatible device consisting of a cylindrical stainless-steel mesh. The device was implanted 40 days prior to islet transplantation to allow embedding by connective tissue and neovascularization. Reversal of diabetes and glycemic control was monitored after islet transplantation. Results. Syngeneic islets transplanted into a SC, neovascularized device restored euglycemia and sustained function long-term. Removal of graft-bearing devices resulted in hyperglycemia. Explanted grafts showed preserved islets and intense vascular networks. Conclusions. Ease of implantation, biocompatibility, and ability to maintain long-term graft function support the potential of our implantable device for cellular-based reparative therapies.

Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis

Background. Primary nonfunction resulting in immediate graft loss is responsible for the failure of a large number of islet transplants. Evidence is accumulating to single out endotoxin contamination of the various reagents needed for islet isolation as a major cause of early graft loss. Methods. Islets isolated with endotoxin-containing (400 endotoxin units/ml) collagenase type V and “endotoxin-free” (3.1 endotoxin units/ml) Liberase™ were compared. Graft function was assessed using a syngeneic murine model of marginal islet mass transplantation. Pro-inflammatory cytokine production by islets was measured by ELISA in culture supernatants, and quantitative reverse transcriptase-PCR. Islet cell apoptosis was measured using the annexin assay. Results. Graft function was significantly delayed when islets were isolated with endotoxin-containing collagenase. Addition of endotoxin to the Liberase™ solution similarly delayed graft function. After 18 hr in culture, collagenase-isolated islets released higher amounts of proinflammatory cytokines compared with Liberase™-isolated islets (interleukin-6: 2185±1174 pg/ml vs. 520±201 pg/ml; tumor necrosis factor-&agr;: 304±298 pg/ml vs. 0; IL-1&bgr;: 12.5 pg/ml±12.5 vs. 0). This observation correlated with higher cytokine mRNA expression in collagenase-isolated islets. The percentage of apoptotic islet cells immediately after isolation was 17.2%±9.4 in collagenase-isolated islets and 7.1%±2.1 in Liberase™-isolated islets. Conclusions. We propose that endotoxin contamination is the primum movens of a chain of events that involves intra-islet cytokine production and release and islet cell apoptosis, and endotoxin contamination can ultimately lead to primary nonfunction in vivo. This emphasizes the fact that using endotoxin-free reagents during isolation is a key factor for successful islet transplantation.

Rescue purification maximizes the use of human islet preparations for transplantation

The relative inefficiency of the islet purification process may hamper obtaining enough islets for transplantation even with adequate pre‐purification counts. In this study, we determined the effect of an additional purification step on total islet yields and pancreas utilization at our center.

Rapamycin impairs in vivo proliferation of islet beta-cells

Background. Progressive graft dysfunction is commonly observed in recipients of islet allografts treated with high doses of rapamycin. This study aimed at evaluating the effect of rapamycin on pancreatic islet cell proliferation in vivo. Methods. The murine pregnancy model was utilized, since a high rate of &bgr;-cell proliferation occurs in a well-defined time frame. Rapamycin (0.2 mg/kg/day) was given to C57BL/6 mice for 5–7 days starting on day 7.5 of pregnancy. Cell proliferation was evaluated by detection of bromodeoxyuridine incorporation by immunohistochemistry. Results. Pregnancy led to increased &bgr;-cell proliferation and islet yield with skewing in islet size distribution as well as higher pancreatic insulin content, when compared to that of nonpregnant females. These effects of pregnancy on &bgr;-cell proliferation and mass were significantly blunted by rapamycin treatment. Minimal effect of rapamycin was observed on islet function both in vivo and in vitro. Rapamycin treatment of islets in vitro resulted in reduced p70s6k phosphorylation, which was paralleled by increased ERK1/2 phosphorylation. Conclusions. Rapamycin treatment reduces the rate of &bgr;-cell proliferation in vivo. This phenomenon may contribute to impair &bgr;-cell renewal in transplanted patients and to the progressive dysfunction observed in islet graft recipients.

ATP-gated P2X3 receptors constitute a positive autocrine signal for insulin release in the human pancreatic β cell

Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human β cells sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca2+ concentration, [Ca2+]i, and hormone release in vitro, we show that human β cells express ionotropic ATP receptors of the P2X3 type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X3 receptors in the β-cell plasma membrane, resulting in increased [Ca2+]i and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the β cells secretory machinery. This may explain how the human pancreatic β cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.

Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic β-cells

Abstract Transplantation of islets is becoming an established method for treating type 1 diabetes. However, viability of islets is greatly affected by necrosis/apoptosis induced by oxidative stress and other insults during isolation and subsequent in vitro culture. Expression of cytoprotective proteins, such as heme oxygenase-1 (HO-1), reduces the deleterious effects of oxidative stress in transplantable islets. We have generated a fusion protein composed of HO-1 and TAT protein transduction domain (TAT/PTD), an 11-aa cell penetrating peptide from the human immunodeficiency virus TAT protein. Transduction of TAT/PTD–HO-1 to insulin-producing cells protects against TNF-α-mediated cytotoxicity. TAT/PTD–HO-1 transduction to islets does not impair islet physiology, as assessed by reversion of chemically induced diabetes in immunodeficient mice. Finally, we report that transduction of HO-1 fusion protein into islets improves islet viability in culture. This approach might have a positive impact on the availability of islets for transplantation.

Device design and materials optimization of conformal coating for islets of Langerhans

Significance Cell encapsulation with biocompatible and permeable hydrogels may allow transplantation without immunosuppression. As an alternative to standard microencapsulation approaches that create single-sized capsules around cell clusters of different sizes, we have designed and optimized a novel approach for conformal coating of islets of Langerhans, resulting in thin, complete, and uniform coatings of similar thickness on differently sized islets. Coated islets exhibited no delay in glucose-stimulated insulin release or loss of function during culture, which is often observed with naked islets. The conformal coating reduces transplant volume relative to traditional encapsulation approaches. When transplanted in syngeneic diabetic mice, conformally coated islets restored and maintained euglycemia for more than 100 d with no foreign body reaction and normal revascularization. Encapsulation of islets of Langerhans may represent a way to transplant islets in the absence of immunosuppression. Traditional methods for encapsulation lead to diffusional limitations imposed by the size of the capsules (600–1,000 μm in diameter), which results in core hypoxia and delayed insulin secretion in response to glucose. Moreover, the large volume of encapsulated cells does not allow implantation in sites that might be more favorable to islet cell engraftment. To address these issues, we have developed an encapsulation method that allows conformal coating of islets through microfluidics and minimizes capsule size and graft volume. In this method, capsule thickness, rather than capsule diameter, is constant and tightly defined by the microdevice geometry and the rheological properties of the immiscible fluids used for encapsulation within the microfluidic system. We have optimized the method both computationally and experimentally, and found that conformal coating allows for complete encapsulation of islets with a thin (a few tens of micrometers) continuous layer of hydrogel. Both in vitro and in vivo in syngeneic murine models of islet transplantation, the function of conformally coated islets was not compromised by encapsulation and was comparable to that of unencapsulated islets. We have further demonstrated that the structural support conferred by the coating materials protected islets from the loss of function experienced by uncoated islets during ex vivo culture.