The influence of intraperitoneal transplantation of free and encapsulated Langerhans islets on the second set phenomenon.

PubMed

Orłowski, Tadeusz; Godlewska, Ewa; Mościcka, Maria; Sitarek, Elzbieta

2003-12-01

To protect the allografts or xenografts against transplant rejection special semipermeable membranes are applied. So far, there are only a few studies on the influence of an immunoisolated graft on the recipient immune system. Therefore, the possibility that an intraperitoneally grafted alginate/poly L-lysine/alginate (APA) coated pancreatic islets graft can effectively sensitize the recipient and provoke second set phenomenon was studied. C3H male mice and male WAG rats were used as donors of full-thickness skin and of free or encapsulated islet intraperitoneal grafts. Male BALB/c mice served as recipients. Skin grafts were performed following the method of Billingham and Medawar. The length of the second skin graft survival time served as the criterion for the sensitizing capacity of the primary graft. APA encapsulation of islets delayed but has not prevented the development of the second set phenomenon. However, the second skin graft rejection time was significantly longer after grafting of encapsulated islets than after free islets transplantation. APA microencapsulation of intraperitoneally transplanted islets delayed but did not prevent the development of the second set phenomenon. Encapsulation does not ensure complete immunoisolation, but only creates "an artificially immunoprivileged site of transplantation."

Delayed revascularization of islets after transplantation by IL-6 blockade in pig to non-human primate islet xenotransplantation model.

PubMed

Min, Byoung-Hoon; Shin, Jun-Seop; Kim, Jong-Min; Kang, Seong-Jun; Kim, Hyun-Je; Yoon, Il-Hee; Park, Su-Kyoung; Choi, Ji-Won; Lee, Min-Suk; Park, Chung-Gyu

2018-01-01

Pancreatic islet transplantation is currently proven as a promising treatment for type 1 diabetes patients with labile glycemic control and severe hypoglycemia unawareness. Upon islet transplantation, revascularization is essential for proper functioning of the transplanted islets. As IL-6 is important for endothelial cell survival and systemic inflammation related to xenograft, the effect of IL-6 receptor antagonist, tocilizumab, on revascularization of the transplanted islets was examined in pig to non-human primate islet xenotransplantation model. Also, the endothelial cell origin in a new vessel of the transplanted pig islets was determined. Pig islets were isolated from designated pathogen-free (DPF) SNU miniature pigs and transplanted via portal vein into five streptozotocin-induced diabetic monkeys. One group (n = 2, basal group) was treated with anti-thymoglobulin (ATG), anti-CD40 antibody (2C10R4), sirolimus, and tacrolimus, and the other group was additionally given tocilizumab on top of basal immunosuppression (n = 3, Tocilizumab group). To confirm IL-6 blocking effect, C-reactive protein (CRP) levels and serum IL-6 concentration were measured. Scheduled biopsy of the margin of the posterior segment right lobe inferior of the liver was performed at 3 weeks after transplantation to assess the degree of revascularization of the transplanted islets. Immunohistochemical staining using anti-insulin, anti-CD31 antibodies, and lectin IB4 was conducted to find the origin of endothelial cells in the islet graft. CRP significantly increased at 1~2 days after transplantation in Basal group, but not in Tocilizumab group, and higher serum IL-6 concentration was measured in latter group, showing the biological potency of tocilizumab. In Basal group, well-developed endothelial cells were observed on the peri- and intraislet area, whereas the number of CD31 + cells in the intraislet space was significantly reduced in Tocilizumab group. Finally, new endothelial cells in the pig islet graft were positive for CD31, but not for lectin IB4, suggesting that they are originated from the recipient monkey. Our results demonstrated that tocilizumab can delay revascularization of the transplanted islet, although this effect had no significant correlation to the overall islet graft survival. In the pig to NHP islet xenotransplantation model, the endothelial cells from recipient monkey form new blood vessels in and around pig islets. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Maintenance of Normoglycemia in Diabetic Mice by Subcutaneous Xenografts of Encapsulated Islets

NASA Astrophysics Data System (ADS)

Lacy, Paul E.; Hegre, Orion D.; Gerasimidi-Vazeou, Andriani; Gentile, Frank T.; Dionne, Keith E.

1991-12-01

The goal of islet transplantation in human diabetes is to maintain the islet grafts in the recipients without the use of immunosuppression. One approach is to encapsulate the donor islets in permselective membranes. Hollow fibers fabricated from an acrylic copolymer were used to encapsulate small numbers of rat islets that were immobilized in an alginate hydrogel for transplantation in diabetic mice. The fibers were biocompatible, prevented rejection, and maintained normoglycemia when transplanted intraperitoneally; hyperglycemia returned when the fibers were removed at 60 days. Normoglycemia was also maintained by subcutaneous implants that had an appropriately constructed outer surface on the fibers.

Human beta-cell precursors mature into functional insulin-producing cells in an immunoisolation device: implications for diabetes cell therapies.

PubMed

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y; Geron, Ifat; Strongin, Alex Y; Itkin-Ansari, Pamela

2009-04-15

Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human beta-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human beta-cells and their progenitors and (2) the engraftment of encapsulated murine beta-cells in allo- and autoimmune settings. Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary beta-cells ameliorated diabetes without stimulating a detectable T-cell response. We demonstrate for the first time that human beta-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of beta-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells.

Survival of Free and Encapsulated Human and Rat Islet Xenografts Transplanted into the Mouse Bone Marrow

PubMed Central

Meier, Raphael P. H.; Seebach, Jörg D.; Morel, Philippe; Mahou, Redouan; Borot, Sophie; Giovannoni, Laurianne; Parnaud, Geraldine; Montanari, Elisa; Bosco, Domenico; Wandrey, Christine; Berney, Thierry; Bühler, Leo H.; Muller, Yannick D.

2014-01-01

Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation. PMID:24625569

Islet Transplantation and Encapsulation: An Update on Recent Developments

PubMed Central

Vaithilingam, Vijayaganapathy; Tuch, Bernard E.

2011-01-01

Human islet transplantation can provide good glycemic control in diabetic recipients without exogenous insulin. However, a major factor limiting its application is the recipient's need to adhere to life-long immunosuppression, something that has serious side effects. Microencapsulating human islets is a strategy that should prevent rejection of the grafted tissue without the need for anti-rejection drugs. Despite promising studies in various animal models, the encapsulated human islets so far have not made an impact in the clinical setting. Many non-immunological and immunological factors such as biocompatibility, reduced immunoprotection, hypoxia, pericapsular fibrotic overgrowth, effects of the encapsulation process and post-transplant inflammation hamper the successful application of this promising technology. In this review, strategies are discussed to overcome the above-mentioned factors and to enhance the survival and function of encapsulated insulin-producing cells, whether in islets or surrogate β-cells. Studies at our center show that barium alginate microcapsules are biocompatible in rodents, but not in humans, raising concerns over the use of rodents to predict outcomes. Studies at our center also show that the encapsulation process had little or no effect on the cellular transcriptome of human islets and on their ability to function either in vitro or in vivo. New approaches incorporating further modifications to the microcapsule surface to prevent fibrotic overgrowth are vital, if encapsulated human islets or β-cell surrogates are to become a viable therapy option for type 1 diabetes in humans. PMID:21720673

A silk-based encapsulation platform for pancreatic islet transplantation improves islet function in vivo.

PubMed

Hamilton, Diana C; Shih, Hank H; Schubert, Richard A; Michie, Sara A; Staats, Paul N; Kaplan, David L; Fontaine, Magali J

2017-03-01

The success of pancreatic islet (PI) transplantation is challenged by PI functional damage during the peritransplantation period. A silk-based encapsulation platform including mesenchymal stromal cells (MSCs) was evaluated for islet cell delivery in vivo. Islet equivalents (IEQs) were transplanted into the epididymal fat pads of mice with streptozotocin-induced diabetes. Three PI combinations were tested: (A) co-encapsulated in silk with MSCs; (b) encapsulated in silk alone; or (c) pelleted. Blood glucose levels were monitored and intraperitoneal glucose tolerance test (IPGTT) was performed upon return to euglycaemia. Grafts were removed for histology and cytokine content analysis. Mice with PI grafts in silk showed a prompt return to euglycaemia. IPGTT was significantly improved with PI in silk with MSCs, compared to PI in silk alone or pelleted. Both Th 1 and Th 2 cytokines were increased in PI grafts in silk, but Th 1 cytokines were decreased significantly with PI and MSC co-encapsulation. Histological analysis showed osteogenesis and chondrogenesis in the silk grafts containing MSCs. Future studies will evaluate MSC stability and function in vivo and improve silk biocompatibility for applications in islet transplantation. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

Human β-cell Precursors Mature Into Functional Insulin-producing Cells in an Immunoisolation Device: Implications for Diabetes Cell Therapies

PubMed Central

Lee, Seung-Hee; Hao, Ergeng; Savinov, Alexei Y.; Geron, Ifat; Strongin, Alex Y.; Itkin-Ansari, Pamela

2009-01-01

Background Islet transplantation is limited by the need for chronic immunosuppression and the paucity of donor tissue. As new sources of human β-cells are developed (e.g., stem cell-derived tissue), transplanting them in a durable device could obviate the need for immunosuppression, while also protecting the patient from any risk of tumorigenicity. Here, we studied (1) the survival and function of encapsulated human β-cells and their progenitors and (2) the engraftment of encapsulated murine β-cells in allo- and autoimmune settings. Methods Human islets and human fetal pancreatic islet-like cell clusters were encapsulated in polytetrafluorethylene devices (TheraCyte) and transplanted into immunodeficient mice. Graft survival and function was measured by immunohistochemistry, circulating human C-peptide levels, and blood glucose levels. Bioluminescent imaging was used to monitor encapsulated neonatal murine islets. Results Encapsulated human islet-like cell clusters survived, replicated, and acquired a level of glucose responsive insulin secretion sufficient to ameliorate hyperglycemia in diabetic mice. Bioluminescent imaging of encapsulated murine neonatal islets revealed a dynamic process of cell death followed by regrowth, resulting in robust long-term allograft survival. Further, in the non-obese diabetic (NOD) mouse model of type I diabetes, encapsulated primary β-cells ameliorated diabetes without stimulating a detectable T-cell response. Conclusions We demonstrate for the first time that human β-cells function is compatible with encapsulation in a durable, immunoprotective device. Moreover, our study suggests that encapsulation of β-cells before terminal differentiation will be a successful approach for new cell-based therapies for diabetes, such as those derived from stem cells. PMID:19352116

Enhanced function of immuno-isolated islets in diabetes therapy by co-encapsulation with an anti-inflammatory drug

PubMed Central

Dang, Tram T.; Thai, Anh V.; Cohen, Joshua; Slosberg, Jeremy E.; Siniakowicz, Karolina; Doloff, Joshua C.; Ma, Minglin; Hollister-Lock, Jennifer; Tang, Katherine; Gu, Zhen; Cheng, Hao; Weir, Gordon C.; Langer, Robert; Anderson, Daniel G.

2013-01-01

Immuno-isolation of islets has the potential to enable the replacement of pancreatic function in diabetic patients. However, host response to the encapsulated islets frequently leads to fibrotic overgrowth with subsequent impairment of the transplanted grafts. Here, we identified and incorporated anti-inflammatory agents into islet-containing microcapsules to address this challenge. In vivo subcutaneous screening of 16 small molecule anti-inflammatory drugs was performed to identify promising compounds that could minimize the formation of fibrotic cell layers. Using parallel non-invasive fluorescent and bioluminescent imaging, we identified dexamethasone and curcumin as the most effective drugs in inhibiting the activities of inflammatory proteases and reactive oxygen species in the host response to subcutaneously injected biomaterials. Next, we demonstrated that co-encapsulating curcumin with pancreatic rat islets in alginate microcapsules reduced fibrotic overgrowth and improved glycemic control in a mouse model of chemically-induced type I diabetes. These results showed that localized administration of anti-inflammatory drug can improve the longevity of encapsulated islets and may facilitate the translation of this technology towards a long-term cure for type I diabetes. PMID:23660251

Silicon nanopore membrane (SNM) for islet encapsulation and immunoisolation under convective transport

NASA Astrophysics Data System (ADS)

Song, Shang; Faleo, Gaetano; Yeung, Raymond; Kant, Rishi; Posselt, Andrew M.; Desai, Tejal A.; Tang, Qizhi; Roy, Shuvo

2016-03-01

Problems associated with islet transplantation for Type 1 Diabetes (T1D) such as shortage of donor cells, use of immunosuppressive drugs remain as major challenges. Immune isolation using encapsulation may circumvent the use of immunosuppressants and prolong the longevity of transplanted islets. The encapsulating membrane must block the passage of host’s immune components while providing sufficient exchange of glucose, insulin and other small molecules. We report the development and characterization of a new generation of semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM), designed with approximately 7 nm-wide slit-pores to provide middle molecule selectivity by limiting passage of pro-inflammatory cytokines. Moreover, the use of convective transport with a pressure differential across the SNM overcomes the mass transfer limitations associated with diffusion through nanometer-scale pores. The SNM exhibited a hydraulic permeability of 130 ml/hr/m2/mmHg, which is more than 3 fold greater than existing polymer membranes. Analysis of sieving coefficients revealed 80% reduction in cytokines passage through SNM under convective transport. SNM protected encapsulated islets from infiltrating cytokines and retained islet viability over 6 hours and remained responsive to changes in glucose levels unlike non-encapsulated controls. Together, these data demonstrate the novel membrane exhibiting unprecedented hydraulic permeability and immune-protection for islet transplantation therapy.

Characterisation of the Xenogeneic Immune Response to Microencapsulated Fetal Pig Islet-Like Cell Clusters Transplanted into Immunocompetent C57BL/6 Mice

PubMed Central

Ratnapala, Sabina; Foster, Jayne; Vaghjiani, Vijesh; Manuelpillai, Ursula; Tuch, Bernard E.

2013-01-01

Xenotransplantation of microencapsulated fetal pig islet-like cell clusters (FP ICCs) offers a potential cellular therapy for type 1 diabetes. Although microcapsules prevent direct contact of the host immune system with the xenografted tissue, poor graft survival is still an issue. This study aimed to characterise the nature of the host immune cells present on the engrafted microcapsules and effects on encapsulated FP ICCs that were transplanted into immunocompetent mice. Encapsulated FP ICCs were transplanted into the peritoneal cavity of C57BL/6 mice. Grafts retrieved at days 1, 3, 7, 14 and 21 post-transplantation were analysed for pericapsular fibrotic overgrowth (PFO), cell viability, intragraft porcine gene expression, macrophages, myofibroblasts and intraperitoneal murine cytokines. Graft function was assessed ex vivo by insulin secretion studies. Xenogeneic immune response to encapsulated FP ICCs was associated with enhanced intragraft mRNA expression of porcine antigens MIP-1α, IL-8, HMGB1 and HSP90 seen within the first two weeks post-transplantation. This was associated with the recruitment of host macrophages, infiltration of myofibroblasts and collagen deposition leading to PFO which was evident from day 7 post-transplantation. This was accompanied by a decrease in cell viability and loss of FP ICC architecture. The only pro-inflammatory cytokine detected in the murine peritoneal flushing was TNF-α with levels peaking at day 7 post transplantation. This correlated with the onset of PFO at day 7 implying activated macrophages as its source. The anti-inflammatory cytokines detected were IL-5 and IL-4 with levels peaking at days 1 and 7, respectively. Porcine C-peptide was undetectable at all time points post-transplantation. PFO was absent and murine intraperitoneal cytokines were undetectable when empty microcapsules were transplanted. In conclusion, this study demonstrated that the macrophages are direct effectors of the xenogeneic immune response to encapsulated FP ICCs leading to PFO mediated by a combination of both pro- and anti-inflammatory cytokines. PMID:23554983

Long-term Efficacy and Biocompatibility of Encapsulated Islet Transplantation With Chitosan-Coated Alginate Capsules in Mice and Canine Models of Diabetes.

PubMed

Yang, Hae Kyung; Ham, Dong-Sik; Park, Heon-Seok; Rhee, Marie; You, Young Hye; Kim, Min Jung; Shin, Juyoung; Kim, On-You; Khang, Gilson; Hong, Tae Ho; Kim, Ji-Won; Lee, Seung-Hwan; Cho, Jae-Hyoung; Yoon, Kun-Ho

2016-02-01

Clinical application of encapsulated islet transplantation is hindered by low biocompatibility of capsules leading to pericapsular fibrosis and decreased islet viability. To improve biocompatibility, we designed a novel chitosan-coated alginate capsules and compared them to uncoated alginate capsules. Alginate capsules were formed by crosslinking with BaCl2, then they were suspended in chitosan solution for 10 minutes at pH 4.5. Xenogeneic islet transplantation, using encapsulated porcine islets in 1,3-galactosyltransferase knockout mice, and allogeneic islet transplantation, using encapsulated canine islets in beagles, were performed without immunosuppressants. The chitosan-alginate capsules showed similar pore size, islet viability, and insulin secretory function compared to alginate capsules, in vitro. Xenogeneic transplantation of chitosan-alginate capsules demonstrated a trend toward superior graft survival (P = 0.07) with significantly less pericapsular fibrosis (cell adhesion score: 3.77 ± 0.41 vs 8.08 ± 0.05; P < 0.001) compared to that of alginate capsules up to 1 year after transplantation. Allogeneic transplantation of chitosan-alginate capsules normalized the blood glucose level up to 1 year with little evidence of pericapsular fibrotic overgrowth on graft explantation. The efficacy and biocompatibility of chitosan-alginate capsules were demonstrated in xenogeneic and allogeneic islet transplantations using small and large animal models of diabetes. This capsule might be a potential candidate applicable in the treatment of type 1 diabetes mellitus patients, and further studies in nonhuman primates are required.

Design of a vascularized synthetic poly(ethylene glycol) macroencapsulation device for islet transplantation.

PubMed

Weaver, Jessica D; Headen, Devon M; Hunckler, Michael D; Coronel, Maria M; Stabler, Cherie L; García, Andrés J

2018-07-01

The use of immunoisolating macrodevices in islet transplantation confers the benefit of safety and translatability by containing transplanted cells within a single retrievable device. To date, there has been limited development and characterization of synthetic poly(ethylene glycol) (PEG)-based hydrogel macrodevices for islet encapsulation and transplantation. Herein, we describe a two-component synthetic PEG hydrogel macrodevice system, designed for islet delivery to an extrahepatic islet transplant site, consisting of a hydrogel core cross-linked with a non-degradable PEG dithiol and a vasculogenic outer layer cross-linked with a proteolytically sensitive peptide to promote degradation and enhance localized vascularization. Synthetic PEG macrodevices exhibited equivalent passive molecular transport to traditional microencapsulation materials (e.g., alginate) and long-term stability in the presence of proteases in vitro and in vivo, out to 14 weeks in rats. Encapsulated islets demonstrated high viability within the device in vitro and the incorporation of RGD adhesive peptides within the islet encapsulating PEG hydrogel improved insulin responsiveness to a glucose challenge. In vivo, the implementation of a vasculogenic, degradable hydrogel layer at the outer interface of the macrodevice enhanced vascular density within the rat omentum transplant site, resulting in improved encapsulated islet viability in a syngeneic diabetic rat model. These results highlight the benefits of the facile PEG platform to provide controlled presentation of islet-supportive ligands, as well as degradable interfaces for the promotion of engraftment and overall graft efficacy. Copyright © 2018 Elsevier Ltd. All rights reserved.

Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment.

PubMed

Sabek, Omaima M; Farina, Marco; Fraga, Daniel W; Afshar, Solmaz; Ballerini, Andrea; Filgueira, Carly S; Thekkedath, Usha R; Grattoni, Alessandro; Gaber, A Osama

2016-01-01

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow-derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates' survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland-islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy.

Three-dimensional printed polymeric system to encapsulate human mesenchymal stem cells differentiated into islet-like insulin-producing aggregates for diabetes treatment

PubMed Central

Sabek, Omaima M; Farina, Marco; Fraga, Daniel W; Afshar, Solmaz; Ballerini, Andrea; Filgueira, Carly S; Thekkedath, Usha R; Grattoni, Alessandro; Gaber, A Osama

2016-01-01

Diabetes is one of the most prevalent, costly, and debilitating diseases in the world. Pancreas and islet transplants have shown success in re-establishing glucose control and reversing diabetic complications. However, both are limited by donor availability, need for continuous immunosuppression, loss of transplanted tissue due to dispersion, and lack of vascularization. To overcome the limitations of poor islet availability, here, we investigate the potential of bone marrow–derived mesenchymal stem cells differentiated into islet-like insulin-producing aggregates. Islet-like insulin-producing aggregates, characterized by gene expression, are shown to be similar to pancreatic islets and display positive immunostaining for insulin and glucagon. To address the limits of current encapsulation systems, we developed a novel three-dimensional printed, scalable, and potentially refillable polymeric construct (nanogland) to support islet-like insulin-producing aggregates’ survival and function in the host body. In vitro studies showed that encapsulated islet-like insulin-producing aggregates maintained viability and function, producing steady levels of insulin for at least 4 weeks. Nanogland—islet-like insulin-producing aggregate technology here investigated as a proof of concept holds potential as an effective and innovative approach for diabetes cell therapy. PMID:27152147

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate and porcine origin

PubMed Central

Mueller, Kate R; Balamurugan, A.N.; Cline, Gary W; Pongratz, Rebecca L; Hooper, Rebecca L; Weegman, Bradley P; Kitzmann, Jennifer P; Taylor, Michael J; Graham, Melanie L; Schuurman, Henk-Jan; Papas, Klearchos K

2014-01-01

Background Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remains a critical issue. Methods Islets isolated from human (n=3), NHP (n=2), adult pig (AP, n=3) and juvenile pig (JP, n=3) pancreata were perifused with medium at basal glucose (2.5mM) followed by high glucose (16.7mM) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Results NHP islets exhibited GSIS 3-fold higher than human islets, while AP and JP islets exhibited GSIS 1/3 and 1/16 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/3 of human islets. Conclusion Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for human, AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation which may be substantially higher than that required for humans. Finally, porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets. PMID:23384163

Differences in glucose-stimulated insulin secretion in vitro of islets from human, nonhuman primate, and porcine origin.

PubMed

Mueller, Kate R; Balamurugan, A N; Cline, Gary W; Pongratz, Rebecca L; Hooper, Rebecca L; Weegman, Bradley P; Kitzmann, Jennifer P; Taylor, Michael J; Graham, Melanie L; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

Porcine islet xenotransplantation is considered a potential cell-based therapy for type 1 diabetes. It is currently being evaluated in diabetic nonhuman primates (NHP) to assess safety and efficacy of the islet product. However, due to a variety of distinct differences between the respective species, including the insulin secretory characteristics of islets, the suitability and predictive value of the preclinical model in the extrapolation to the clinical setting remain a critical issue. Islets isolated from human (n = 3), NHP (n = 2), adult pig (AP, n = 3), and juvenile pig (JP, n = 4) pancreata were perifused with medium at basal glucose (2.5 mm) followed by high glucose (16.7 mm) concentrations. The total glucose-stimulated insulin secretion (GSIS) was calculated from generated insulin secretion profiles. Nonhuman primate islets exhibited GSIS 3-fold higher than AP islets, while AP and JP islets exhibited GSIS 1/3 and 1/30 of human islets, respectively. The insulin content of NHP and AP islets was similar to that of human islets, whereas that of JP islets was 1/5 of human islets. Despite the fact that human, NHP, and AP islets contain similar amounts of insulin, the much higher GSIS for NHP islets than for AP and JP islets suggests the need for increased dosing of islets from JP and AP in pig-to-NHP transplantation. Porcine islet xenotransplantation to humans may require significantly higher dosing given the lower GSIS of AP islets compared to human islets. © 2013 John Wiley & Sons A/S.

Enhanced function of immuno-isolated islets in diabetes therapy by co-encapsulation with an anti-inflammatory drug.

PubMed

Dang, Tram T; Thai, Anh V; Cohen, Joshua; Slosberg, Jeremy E; Siniakowicz, Karolina; Doloff, Joshua C; Ma, Minglin; Hollister-Lock, Jennifer; Tang, Katherine M; Gu, Zhen; Cheng, Hao; Weir, Gordon C; Langer, Robert; Anderson, Daniel G

2013-07-01

Immuno-isolation of islets has the potential to enable the replacement of pancreatic function in diabetic patients. However, host response to the encapsulated islets frequently leads to fibrotic overgrowth with subsequent impairment of the transplanted grafts. Here, we identified and incorporated anti-inflammatory agents into islet-containing microcapsules to address this challenge. In vivo subcutaneous screening of 16 small molecule anti-inflammatory drugs was performed to identify promising compounds that could minimize the formation of fibrotic cell layers. Using parallel non-invasive fluorescent and bioluminescent imaging, we identified dexamethasone and curcumin as the most effective drugs in inhibiting the activities of inflammatory proteases and reactive oxygen species in the host response to subcutaneously injected biomaterials. Next, we demonstrated that co-encapsulating curcumin with pancreatic rat islets in alginate microcapsules reduced fibrotic overgrowth and improved glycemic control in a mouse model of chemically-induced type I diabetes. These results showed that localized administration of anti-inflammatory drug can improve the longevity of encapsulated islets and may facilitate the translation of this technology toward a long-term cure for type I diabetes. Published by Elsevier Ltd.

The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs.

PubMed

Krickhahn, Mareike; Bühler, Christoph; Meyer, Thomas; Thiede, Arnulf; Ulrichs, Karin

2002-01-01

Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.

Injectable Polyethylene Glycol Hydrogel for Islet Encapsulation: an in vitro and in vivo Characterization

PubMed Central

Knobeloch, Tracy; Abadi, Sakineh Esmaeili Mohsen; Bruns, Joseph; Zustiak, Silviya Petrova; Kwon, Guim

2017-01-01

An injection of hydrogel-encapsulated islets that controls blood glucose levels over long term would provide a much needed alternative treatment for type 1 diabetes mellitus (T1DM). To this end, we tested the feasibility of using an injectable polyethylene glycol (PEG) hydrogel as a scaffold for islet encapsulation. Encapsulated islets cultured in vitro for 6 days showed excellent cell viability and released insulin with higher basal and stimulated insulin secretion than control islets. Host responses to PEG hydrogels were studied by injecting PEG hydrogels (no treatment and vehicle controls used) into the peritoneal cavities of B6D2F1 mice and monitoring alterations in body weight, food and water intake, and blood glucose levels. After 2 weeks, peritoneal cavity cells were harvested, followed by hydrogel retrieval, and extraction of spleens. Body weights, food and water intake, and blood glucose levels were unaltered in mice injected with hydrogels compared to no treatment and vehicle-injected control mice. Frozen sections of a hydrogel showed the presence of tissues and small number of immune cells surrounding the hydrogel but no cell infiltration into the hydrogel bulk. Spleen sizes were not significantly different under the experimental conditions. Peritoneal cavity cells were slightly higher in mice injected with hydrogels compared to control mice but no statistical difference between vehicle- and hydrogel-injected mice was noted. As an in vivo feasibility study, streptozotocin-induced diabetic mice were injected with vehicle or hydrogels containing 50 islets each into two sites, the peritoneal cavity and a subcutaneous site on the back. Transient control of blood glucose levels were observed in mice injected with hydrogels containing islets. In summary, we developed an injectable PEG hydrogel that supported islet function and survival in vitro and in vivo and elicited only a mild host response. Our work illustrates the feasibility of using injectable PEG hydrogels for islet encapsulation. PMID:29527325

Failure of transplantation tolerance induction by autologous regulatory T cells in the pig-to-non-human primate islet xenotransplantation model.

PubMed

Shin, Jun-Seop; Min, Byoung-Hoon; Kim, Jong-Min; Kim, Jung-Sik; Yoon, Il Hee; Kim, Hyun Je; Kim, Yong-Hee; Jang, Jae Yool; Kang, Hee Jung; Lim, Dong-Gyun; Ha, Jongwon; Kim, Sang-Joon; Park, Chung-Gyu

2016-07-01

Islet allotransplantation is a promising way to treat some type 1 diabetic (T1D) patients with frequent hypoglycemic unawareness, and islet xenotransplantation is emerging to overcome the problem of donor organ shortage. Our recent study showing reproducible long-term survival of porcine islets in non-human primates (NHPs) allows us to examine whether autologous regulatory T-cell (Treg) infusion at peri-transplantation period would induce transplantation tolerance in xenotransplantation setting. Two diabetic rhesus monkeys were transplanted with porcine islets from wild-type adult Seoul National University (SNU) miniature pigs with immunosuppression by anti-thymoglobulin (ATG), cobra venom factor, anti-CD154 monoclonal antibody (mAb), and sirolimus. CD4(+) CD25(high) CD127(low) autologous regulatory T cells from the recipients were isolated, ex vivo expanded, and infused at the peri-transplantation period. Blood glucose and porcine C-peptide from the recipients were measured up to 1000 days. Maintenance immunosuppressants including a CD40-CD154 blockade were deliberately discontinued to confirm whether transplantation tolerance was induced by adoptively transferred Tregs. After pig islet transplantation via portal vein, blood glucose levels of diabetic recipients became normalized and maintained over 6 months while in immunosuppressive maintenance with a CD40-CD154 blockade and sirolimus. However, the engrafted pig islets in the long-term period were fully rejected by activated immune cells, particularly T cells, when immunosuppressants were stopped, showing a failure of transplantation tolerance induction by autologous Tregs. Taken together, autologous Tregs infused at the peri-transplantation period failed to induce transplantation tolerance in pig-to-NHP islet xenotransplantation setting. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Immunological Challenges Facing Translation of Alginate Encapsulated Porcine Islet Xenotransplantation to Human Clinical Trials.

PubMed

Krishnan, Rahul; Ko, David; Foster, Clarence E; Liu, Wendy; Smink, A M; de Haan, Bart; De Vos, Paul; Lakey, Jonathan R T

2017-01-01

Transplantation of alginate-encapsulated islets has the potential to treat patients suffering from type I diabetes, a condition characterized by an autoimmune attack against insulin-secreting beta cells. However, there are multiple immunological challenges associated with this procedure, all of which must be adequately addressed prior to translation from trials in small animal and nonhuman primate models to human clinical trials. Principal threats to graft viability include immune-mediated destruction triggered by immunogenic alginate impurities, unfavorable polymer composition and surface characteristics, and release of membrane-permeable antigens, as well as damage associated molecular patterns (DAMPs) by the encapsulated islets themselves. The lack of standardization of significant parameters of bioencapsulation device design and manufacture (i.e., purification protocols, surface-modification grafting techniques, alginate composition modifications) between labs is yet another obstacle that must be overcome before a clinically effective and applicable protocol for encapsulating islets can be implemented. Nonetheless, substantial progress is being made, as is evident from prolonged graft survival times and improved protection from immune-mediated graft destruction reported by various research groups, but also with regard to discoveries of specific pathways involved in explaining observed outcomes. Progress in the latter is essential for a comprehensive understanding of the mechanisms responsible for the varying levels of immunogenicity of certain alginate devices. Successful translation of encapsulated islet transplantation from in vitro and animal model testing to human clinical trials hinges on application of this knowledge of the pathways and interactions which comprise immune-mediated rejection. Thus, this review not only focuses on the different factors contributing to provocation of the immune reaction by encapsulated islets, but also on the defining characteristics of the response itself.

Clinical porcine islet xenotransplantation under comprehensive regulation.

PubMed

Matsumoto, S; Tan, P; Baker, J; Durbin, K; Tomiya, M; Azuma, K; Doi, M; Elliott, R B

2014-01-01

Xenotransplantation with porcine islets is a promising approach to overcome the shortage of human donors. This is the first report of phase 1/2a xenotransplantation study of encapsulated neonatal porcine islets under the current framework of regulations for xenotransplantation in New Zealand. Newborn piglets were anesthetized and bled, and the pancreata were removed with the use of sterile technique and processed. Encapsulated neonatal porcine islets were implanted with the use of laparoscopy into the peritoneal cavity of 14 patients with unstable type 1 diabetes without any immunosuppressive drugs. The patients received encapsulated islets of 5,000 (n = 4; group 1), 10,000 (n = 4; group 2), 15,000 (n = 4; group 3), or 20,000 (n = 2; group 4) islet equivalents per kg body weight. Outcome was determined from adverse event reports, HbA1c, total daily insulin dose, and frequency of unaware hypoglycemic events. To assess graft function, transplant estimated function (TEF) scores were calculated. Sufficient or marginal numbers of encapsulated neonatal porcine islets were transplanted into streptozotocin-induced diabetic B6 mice as an in vivo functional assay. There were 4 serious adverse events, of which 3 were considered to be possibly related to the procedure. Tests for porcine endogenous retrovirus DNA and RNA were all negative. The numbers of unaware hypoglycemia events were reduced after transplantation in all groups. Four of 14 patients attained HbA1c <7% compared with 1 at baseline. The average TEF scores were 0.17, 0.02, -0.01, and 0.08 in groups 1, 2, 3, and 4 respectively. The in vivo study demonstrated that a sufficient number of the transplanted group reversed diabetes with positive porcine C-peptide. Transplantation of encapsulated neonatal porcine islets was safe and was followed by a reduction in unaware hypoglycemia events in unstable type 1 diabetic patients. The mouse in vivo assessment data demonstrated certain graft function. Copyright © 2014 Elsevier Inc. All rights reserved.

Treatment of diabetic rats with encapsulated islets.

PubMed

Sweet, Ian R; Yanay, Ofer; Waldron, Lanaya; Gilbert, Merle; Fuller, Jessica M; Tupling, Terry; Lernmark, Ake; Osborne, William R A

2008-12-01

Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose>350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30-40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats.

Real-time assessment of encapsulated neonatal porcine islets prior to clinical xenotransplantation.

PubMed

Kitzmann, Jennifer P; Law, Lee; Shome, Avik; Muzina, Marija; Elliott, Robert B; Mueller, Kate R; Schuurman, Henk-Jan; Papas, Klearchos K

2012-01-01

Porcine islet transplantation is emerging as an attractive option for the treatment of patients with type 1 diabetes, with the possibility of providing islets of higher and more consistent quality and in larger volumes than available from human pancreata. The use of encapsulated neonatal porcine islets (ENPI) is appealing because it can address islet supply limitations while reducing the need for anti-rejection therapy. Pre-transplant characterization of ENPI viability and potency is an essential component of the production process. We applied the validated assay for oxygen consumption rate normalized for DNA content (OCR/DNA) to characterize ENPI viability. ENPI of low viscosity and high m alginate were prepared according to standard methods and characterized at various culture time points up to 5 weeks. The OCR/DNA (nmol/min·mgDNA ± SEM) of ENPI (235 ± 10, n = 9) was comparable to that of free NPI (255 ± 14, n = 13). After encapsulation, NPI OCR/DNA was sustained over a culture period of up to 5 weeks. The average OCR/DNA of ENPI cultured longer than 9 days was higher than that of freshly encapsulated NPI. This is the first characterization of ENPI by a validated and more sensitive method for product viability. The NPI encapsulation process does not compromise viability as measured by OCR/DNA, and ENPI can be cultured for up to 5 weeks with maintenance of viability. ENPI meet or exceed current adult porcine islet product release criteria (established at the University of Minnesota) for preclinical xenotransplantation in terms of OCR/DNA. © 2012 John Wiley & Sons A/S.

Plasticity and Aggregation of Juvenile Porcine Islets in Modified Culture: Preliminary Observations.

PubMed

Weegman, Bradley P; Taylor, Michael J; Baicu, Simona C; Mueller, Kate; O'brien, Timothy D; Wilson, John; Papas, Klearchos K

2016-10-01

Diabetes is a major health problem worldwide, and there is substantial interest in developing xenogeneic islet transplantation as a potential treatment. The potential to relieve the demand on an inadequate supply of human pancreata is dependent upon the efficiency of techniques for isolating and culturing islets from the source pancreata. Porcine islets are favored for xenotransplantation, but mature pigs (>2 years) present logistic and economic challenges, and young pigs (3-6 months) have not yet proven to be an adequate source. In this study, islets were isolated from 20 juvenile porcine pancreata (~3 months; 25 kg Yorkshire pigs) immediately following procurement or after 24 h of hypothermic machine perfusion (HMP) preservation. The resulting islet preparations were characterized using a battery of tests during culture in silicone rubber membrane flasks. Islet biology assessment included oxygen consumption, insulin secretion, histopathology, and in vivo function. Islet yields were highest from HMP-preserved pancreata (2,242 ± 449 IEQ/g). All preparations comprised a high proportion (>90%) of small islets (<100 μm), and purity was on average 63 ± 6%. Morphologically, islets appeared as clusters on day 0, loosely disaggregated structures at day 1, and transitioned to aggregated structures comprising both exocrine and endocrine cells by day 6. Histopathology confirmed both insulin and glucagon staining in cultures and grafts excised after transplantation in mice. Nuclear staining (Ki-67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 175 ± 16 nmol/min/mg DNA, and physiological function was intact by glucose stimulation after 6-8 days in culture. In vivo function was confirmed with blood glucose control achieved in nearly 50% (8/17) of transplants. Preparation and culture of juvenile porcine islets as a source for islet transplantation require specialized conditions. These immature islets undergo plasticity in culture and form fully functional multicellular structures. Further development of this method for culturing immature porcine islets is expected to generate small pancreatic tissue-derived organoids termed "pancreatites," as a therapeutic product from juvenile pigs for xenotransplantation and diabetes research.

Nanomaterial Solutions for the Protection of Insulin Producing Beta Cells

NASA Astrophysics Data System (ADS)

Atchison, Nicole Ann

Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) to beta cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat beta cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.

Evolution of Islet Transplantation for the Last 30 Years.

PubMed

Farney, Alan C; Sutherland, David E R; Opara, Emmanuel C

2016-01-01

In this article, we will review the changes that have occurred in islet transplantation at the birth of Pancreas 30 years ago. The first attempts at β-cell replacement in humans, pancreas and islet transplantation, were performed in the 1960s and 1970s. Although pancreas transplantation has been an accepted treatment for severe labile diabetes predating the emergence of the journal, allogeneic islet transplantation remains experimental. Current investigations within islet transplantation focus to improve islet function after transplantation. Improving islet viability during isolation, exploring ways to increase engraftment, and protection from the host immune system are some of the goals of these investigative efforts. The major barriers to clinical islet transplantation are shortage of human pancreas, the need for immunosuppression, and the inadequacy of the islet isolation process. It is generally accepted that islet encapsulation is an immunoisolation tool with good potential to address the first 2 of those barriers. We have therefore devoted a major part of this review to the critical factors needed to make it a clinical reality. With improved islet isolation techniques and determination of the best site of engraftment as well as improved encapsulation techniques, we hope that islet transplantation could someday achieve routine clinical use.

Treatment of diabetic rats with encapsulated islets

PubMed Central

Sweet, Ian R; Yanay, Ofer; Waldron, Lanaya; Gilbert, Merle; Fuller, Jessica M; Tupling, Terry; Lernmark, Ake; Osborne, William R A

2008-01-01

Immunoprotection of islets using bioisolator systems permits introduction of allogeneic cells to diabetic patients without the need for immunosuppression. Using TheraCyte™ immunoisolation devices, we investigated two rat models of type 1 diabetes mellitus (T1DM), BB rats and rats made diabetic by streptozotocin (STZ) treatment. We chose to implant islets after the onset of diabetes to mimic the probable treatment of children with T1DM as they are usually diagnosed after disease onset. We encapsulated 1000 rat islets and implanted them subcutaneously (SQ) into diabetic biobreeding (BB) rats and STZ-induced diabetic rats, defined as two or more consecutive days of blood glucose >350 mg/dl. Rats were monitored for weight and blood glucose. Untreated BB rats rapidly lost weight and were euthanized at >20% weight loss that occurred between 4 and 10 days from implantation. For period of 30–40 days following islet implantation weights of treated rats remained steady or increased. Rapid weight loss occurred after surgical removal of devices that contained insulin positive islets. STZ-treated rats that received encapsulated islets showed steady weight gain for up to 130 days, whereas untreated control rats showed steady weight loss that achieved >20% at around 55 days. Although islet implants did not normalize blood glucose, treated rats were apparently healthy and groomed normally. Autologous or allogeneic islets were equally effective in providing treatment. TheraCyte™ devices can sustain islets, protect allogeneic cells from immune attack and provide treatment for diabetic-mediated weight loss in both BB rats and STZ-induced diabetic rats. PMID:18373735

Development of an encapsulated stem cell-based therapy for diabetes.

PubMed

Tomei, Alice Anna; Villa, Chiara; Ricordi, Camillo

2015-01-01

Islet transplantation can treat the most severe cases of type 1 diabetes but it currently requires deceased donor pancreata as an islet source and chronic immunosuppression to prevent rejection and recurrence of autoimmunity. Stem cell-derived insulin-producing cells may address the shortage of organ donors, whereas cell encapsulation may reduce or eliminate the requirement for immunosuppression, minimizing the risks associated with the islet transplantation procedure, and potentially prolonging graft survival. This review focuses on the design principles for immunoisolation devices and on stem cell differentiation into insulin-producing cell products. The reader will gain understanding of the different types of immunoisolation devices and the key parameters that affect the outcome of the encapsulated graft. Progresses in stem cell differentiation towards mature endocrine islet cells, including the most recent clinical trials and the challenges associated with the application of immunoisolation devices designed for primary islets to stem-cell products, are also discussed. Recent advancements in the field of stem cell-derived islet cell products and immunoisolation strategies hold great promise for type 1 diabetes. However, a combination product including both cells and an immunoisolation strategy still needs to be optimized and tested for safety and efficacy.

Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

PubMed

Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome.

Xenografted islet cell clusters from INSLEA29Y transgenic pigs rescue diabetes and prevent immune rejection in humanized mice.

PubMed

Klymiuk, Nikolai; van Buerck, Lelia; Bähr, Andrea; Offers, Monika; Kessler, Barbara; Wuensch, Annegret; Kurome, Mayuko; Thormann, Michael; Lochner, Katharina; Nagashima, Hiroshi; Herbach, Nadja; Wanke, Rüdiger; Seissler, Jochen; Wolf, Eckhard

2012-06-01

Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

Pancreatic Islet Transplantation

MedlinePlus

... long-term use of immunosuppressive medications. For example, one approach is to transplant islets encapsulated with a special ... available in the United States. 2 However, only 1,562 pancreases were ... are pursuing various approaches to solve this shortage of islets, such as ...

Microencapsulated cells as hormone delivery systems.

PubMed

Sun, A M; Goosen, M F; O'Shea, G

1987-01-01

Transplantation of pancreatic islets of Langerhans has been shown to prevent the development of many of the complications associated with diabetes. Transplanted islets, however, are readily rejected by the immune system. The use of artificial membranes to isolate the transplanted islets from the immune system of the host prolongs islet allografts in experimental animals. We have developed a method for encapsulating islets in semipermeable membranes composed of alginate and polylysine. The same technique can be applied to other endocrine cell types. The capsules are 700 to 800 micron in diameter with a hydrogel membrane approximately 4 micron thick. Intraperitoneal allografts of 5 x 10(3) encapsulated islets reversed diabetes in rats for up to 21 months and intact capsules with viable beta cells could be recovered from the recipients. Microencapsulation of endocrine cells for transplantation could potentially be used in the clinical treatment of hormone deficiency diseases.

Glucose metabolism in pigs expressing human genes under an insulin promoter.

PubMed

Wijkstrom, Martin; Bottino, Rita; Iwase, Hayoto; Hara, Hidetaka; Ekser, Burcin; van der Windt, Dirk; Long, Cassandra; Toledo, Frederico G S; Phelps, Carol J; Trucco, Massimo; Cooper, David K C; Ayares, David

2015-01-01

Xenotransplantation of porcine islets can reverse diabetes in non-human primates. The remaining hurdles for clinical application include safe and effective T-cell-directed immunosuppression, but protection against the innate immune system and coagulation dysfunction may be more difficult to achieve. Islet-targeted genetic manipulation of islet-source pigs represents a powerful tool to protect against graft loss. However, whether these genetic alterations would impair islet function is unknown. On a background of α1,3-galactosyltransferase gene-knockout (GTKO)/human (h)CD46, additional genes (hCD39, human tissue factor pathway inhibitor, porcine CTLA4-Ig) were inserted in different combinations under an insulin promoter to promote expression in islets (confirmed by immunofluorescence). Seven pigs were tested for baseline and glucose/arginine-challenged levels of glucose, insulin, C-peptide, and glucagon. This preliminary study did not show definite evidence of β-cell deficiencies, even when three transgenes were expressed under the insulin promoter. Of seven animals, all were normoglycemic at fasting, and five of seven had normal glucose disposal rates after challenge. All animals exhibited insulin, C-peptide, and glucagon responses to both glucose and arginine challenge; however, significant interindividual variation was observed. Multiple islet-targeted transgenic expression was not associated with an overtly detrimental effect on islet function, suggesting that complex genetic constructs designed for islet protection warrants further testing in islet xenotransplantation models. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

PubMed

Smith, Kate E; Kelly, Amy C; Min, Catherine G; Weber, Craig S; McCarthy, Fiona M; Steyn, Leah V; Badarinarayana, Vasudeo; Stanton, J Brett; Kitzmann, Jennifer P; Strop, Peter; Gruessner, Angelika C; Lynch, Ronald M; Limesand, Sean W; Papas, Klearchos K

2017-11-01

Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived β cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high β cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic β cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to β cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and β cell function and leads to increased inflammatory signaling.

Survival of encapsulated islets: More than a membrane story

PubMed Central

Barkai, Uriel; Rotem, Avi; de Vos, Paul

2016-01-01

At present, proven clinical treatments but no cures are available for diabetes, a global epidemic with a huge economic burden. Transplantation of islets of Langerhans by their infusion into vascularized organs is an experimental clinical protocol, the first approach to attain cure. However, it is associated with lifelong use of immunosuppressants. To overcome the need for immunosuppression, islets are encapsulated and separated from the host immune system by a permselective membrane. The lead material for this application is alginate which was tested in many animal models and a few clinical trials. This review discusses all aspects related to the function of transplanted encapsulated islets such as the basic requirements from a permselective membrane (e.g., allowable hydrodynamic radii, implications of the thickness of the membrane and relative electrical charge). Another aspect involves adequate oxygen supply, which is essential for survival/performance of transplanted islets, especially when using large retrievable macro-capsules implanted in poorly oxygenated sites like the subcutis. Notably, islets can survive under low oxygen tension and are physiologically active at > 40 Torr. Surprisingly, when densely crowded, islets are fully functional under hyperoxic pressure of up to 500 Torr (> 300% of atmospheric oxygen tension). The review also addresses an additional category of requirements for optimal performance of transplanted islets, named auxiliary technologies. These include control of inflammation, apoptosis, angiogenesis, and the intra-capsular environment. The review highlights that curing diabetes with a functional bio-artificial pancreas requires optimizing all of these aspects, and that significant advances have already been made in many of them. PMID:27011906

Microfluidic-Based Generation of Size-Controlled, Biofunctionalized Synthetic Polymer Microgels for Cell Encapsulation

PubMed Central

Headen, Devon M.; Aubry, Guillaume; Lu, Hang

2014-01-01

Cell and islet microencapsulation in synthetic hydrogels provide an immunoprotective and cell-supportive microenvironment. A microfluidic strategy for the genaration of biofunctionalized, synthetic microgel particles with precise control over particle size and molecular permeability for cell and protein delivery is presented. These engineered capsules support high cell viability and function of encapsulated human stem cells and islets. PMID:24615922

Microencapsulation of Islets of Langerhans via selective withdrawal to achieve immunoisolation

NASA Astrophysics Data System (ADS)

Wyman, Jason; Dillmore, Shannon; Murphy, William; Garfinkel, Marc; Mrksich, Milan; Nagel, Sidney

2004-03-01

Cohen phet al. [1] described how the selective-withdrawal geometry may be used to microencapsulate particles in thin coats whose thickness is independent of the size of the encapsulated particle. We have applied a modified version of this geometry to the microencapsulation of Islets of Langerhans for the purpose of immunoisolation. The Islets are initially placed in a polymer-containing aqueous solution which is then drawn up into a selective-withdrawal spout. As that spout breaks up, it leaves the Islets coated with the polymer solution. These coats are then photo-crosslinked leaving the Islets encapsulated in a hydrogel coating. This coating provides a semi-permeable membrane which allows for the diffusion of small molecules such as nutrients, glucose, and insulin, but which excludes larger proteins such as antibodies. If one can successfully microencapsulate 10^6 islets in uniform coats such as these, then one may transplant Islets without immuno-suppression as a treatment for Type-I Diabetes. We will discuss preliminary phin vitro results. [1] I. Cohen, H. Li, J. L. Hougland, M. Mrksich, and S. R. Nagel Science 292, 265-267 (2001).

Mechanical characterization and numerical simulation of a subcutaneous implantable 3D printed cell encapsulation system.

PubMed

Adamo, Federica; Farina, Marco; Thekkedath, Usha R; Grattoni, Alessandro; Sesana, Raffaella

2018-06-01

Cell transplantation in bioengineered scaffolds and encapsulation systems has shown great promise in regenerative medicine. Depending on the site of implantation, type of cells and their expected function, these systems are designed to provide cells with a physiological-like environment while providing mechanical support and promoting long-term viability and function of the graft. A minimally invasive 3D printed system termed neovascularized implantable cell homing and encapsulation (NICHE) was developed in polylactic acid for subcutaneous transplantation of endocrine cells, including pancreatic islets. The suitability of the NICHE for long term in vivo deployment is investigated by assessing mechanical behavior of both fresh devices under simulated subcutaneous conditions and NICHE retrieved from subcutaneous implantation in pigs. Both experimental and numerical studies were performed with a focus on validating the constitutive material model used in the numerical analysis for accuracy and reliability. Notably, homogeneous isotropic constitutive material model calibrated by means of uniaxial testing well suited experimental results. The results highlight the long term durability for in vivo applications and the potential applicability of the model to predict the mechanical behavior of similar devices in various physiological settings. Copyright © 2018 Elsevier Ltd. All rights reserved.

Factors influencing the adequacy of microencapsulation of rat pancreatic islets.

PubMed

De Vos, P; De Haan, B; Wolters, G H; Van Schilfgaarde, R

1996-10-15

The observation that only a portion of all alginate-polylysine microcapsules are overgrown after implantation suggests that physical imperfections of individual capsules, rather than the chemical composition of the material applied, are responsible for inducing insufficient biocompatibility and thereby fibrotic overgrowth of those capsules. We recently developed a lectin binding assay that allows for quantifying the portion of inadequately encapsulated islets, and demonstrated that inadequately encapsulated islets induce a fibrotic response associated with graft failure. The present study investigates factors influencing the adequacy of encapsulation of pancreatic islets. We applied our lectin binding assay and found that the number of inadequate, and particularly incomplete, capsules is influenced by the following factors. (1) A capsule diameter of 800 micrometers is associated with a lower percentage of inadequate capsules than smaller (500 micrometers and 600 micrometers) or larger (1800 micrometers) capsules. (2) A high rather than low guluronic acid content of the alginate is associated with a lower percentage of inadequate capsules. This can be explained, at least in part, by smaller ranges of swelling and subsequent shrinkage during the encapsulation procedure. (3) An increase in viscosity caused by applying a higher alginate concentration compensates for a low guluronic acid content. This effect of increased viscosity cannot be explained by a reduced range of swelling and shrinkage during the encapsulation procedure. We conclude that alginates with a high guluronic acid content and a viscosity near the filtration limit are preferable in order to minimize the number of inadequate capsules.

Human embryonic stem cell derived islet progenitors mature inside an encapsulation device without evidence of increased biomass or cell escape.

PubMed

Kirk, Kaitlyn; Hao, Ergeng; Lahmy, Reyhaneh; Itkin-Ansari, Pamela

2014-05-01

There are several challenges to successful implementation of a cell therapy for insulin dependent diabetes derived from human embryonic stem cells (hESC). Among these are development of functional insulin producing cells, a clinical delivery method that eliminates the need for chronic immunosuppression, and assurance that hESC derived tumors do not form in the patient. We and others have shown that encapsulation of cells in a bilaminar device (TheraCyte) provides immunoprotection in rodents and primates. Here we monitored human insulin secretion and employed bioluminescent imaging (BLI) to evaluate the maturation, growth, and containment of encapsulated islet progenitors derived from CyT49 hESC, transplanted into mice. Human insulin was detectable by 7 weeks post-transplant and increased 17-fold over the course of 8 weeks, yet during this period the biomass of encapsulated cells remained constant. Remarkably, by 20 weeks post-transplant encapsulated cells secreted sufficient levels of human insulin to ameliorate alloxan induced diabetes. Further, bioluminescent imaging revealed for the first time that hESCs remained fully contained in encapsulation devices for up to 150 days, the longest period tested. Collectively, the data suggest that encapsulated hESC derived islet progenitors hold great promise as an effective and safe cell replacement therapy for insulin dependent diabetes. Copyright © 2014. Published by Elsevier B.V.

Imaging of Hydrogel Microsphere Structure and Foreign Body Response Based on Endogenous X-Ray Phase Contrast

DOE PAGES

Appel, Alyssa A.; Ibarra, Veronica; Somo, Sami I.; ...

2016-10-31

Transplantation of functional islets encapsulated in stable biomaterials has the potential to cure Type I diabetes. However, the success of these materials requires the ability to understand their stability in vivo. Imaging techniques that enable monitoring of biomaterial performance are critical to further development in the field. In this study, we demonstrate for the first time that X-ray phase contrast (XPC) imaging techniques enable 3D imaging and evaluation of islet volume, alginate hydrogel structure and local soft tissue response. Islets were encapsulated in alginate systems prepared in methods used in clinical trials and implanted in a rodent omentum pouch modelmore » as a treatment for type I diabetes. Microbeads were imaged with XPC prior to implantation and following implantation into an omentum pouch. Islets could be identified within alginate beads and the islet volume quantified. Omental adipose tissue could be distinguished from inflammatory regions resulting from implanted beads. Individual beads and the local encapsulation response were visualized and quantifiable. Measurements were in agreement with histology. The 3D structure of the microbeads could be characterized with XPC and failed beads could also be identified. These results point to the substantial potential of XPC as a tool for imaging biomaterials in small animal models.« less

Imaging of Hydrogel Microsphere Structure and Foreign Body Response Based on Endogenous X-Ray Phase Contrast

DOE Office of Scientific and Technical Information (OSTI.GOV)

Appel, Alyssa A.; Ibarra, Veronica; Somo, Sami I.

Transplantation of functional islets encapsulated in stable biomaterials has the potential to cure Type I diabetes. However, the success of these materials requires the ability to understand their stability in vivo. Imaging techniques that enable monitoring of biomaterial performance are critical to further development in the field. In this study, we demonstrate for the first time that X-ray phase contrast (XPC) imaging techniques enable 3D imaging and evaluation of islet volume, alginate hydrogel structure and local soft tissue response. Islets were encapsulated in alginate systems prepared in methods used in clinical trials and implanted in a rodent omentum pouch modelmore » as a treatment for type I diabetes. Microbeads were imaged with XPC prior to implantation and following implantation into an omentum pouch. Islets could be identified within alginate beads and the islet volume quantified. Omental adipose tissue could be distinguished from inflammatory regions resulting from implanted beads. Individual beads and the local encapsulation response were visualized and quantifiable. Measurements were in agreement with histology. The 3D structure of the microbeads could be characterized with XPC and failed beads could also be identified. These results point to the substantial potential of XPC as a tool for imaging biomaterials in small animal models.« less

The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--chapter 3: Pig islet product manufacturing and release testing.

PubMed

Korbutt, Gregory S

2009-01-01

This chapter provides recommendations on pig islet product manufacturing and release testing to scientific and corporate programs interested in future clinical studies using xenogeneic porcine pancreatic islet cell products for the treatment of type 1 diabetes.To facilitate control of manufacturing as well as reproducibility and consistency of product lots, the manufacturing process, and the manufacturing facility must be in compliance with current Good Manufacturing Practices regulations. Data must be provided to demonstrate that islet products can be consistently prepared that would meet basic lot release requirements. To facilitate product safety: (i) materials used in the manufacturing process, including the pig pancreas, must be free of adventitious agents; (ii) islets must be manufactured using aseptic processing; and (iii) final product must undergo tests for sterility, mycoplasma (if cultured) and endotoxin. Safety specifications for pig islet product release include a negative Gram stain and an endotoxin content of <5.0 EU/kg recipient body weight. Product post-release assessments must include sterility cultures on the final product. Because results for sterility are available only retrospectively, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive for contamination. Product characterization information must address important aspects of lot release testing such as identity/purity (cell composition), quantity [islet equivalents (IE), cell number] and potency (insulin secretory capacity, oxygen consumption rate corrected for DNA or transplant bioassay in immunoincompetent diabetic mice). This information is also critical to demonstrate manufacturing control and product consistency across multiple islet preparations (lots). Providing islet products containing an islet mass sufficient to restore euglycemia in trial participants (>or=10 000 IE/kg) requires pooling of islets from multiple donor pancreata (two to four from adult donors and seven to 10 from neonatal donors). Demonstration of product consistency across products from individual pancreata would warrant release testing to be performed on a sample of the pooled product. As product development and clinical trials advance, the increasingly more detailed specifications of potency assays on adult porcine islet products are expected to be predictive of post-transplant glycemic control. The immaturity of fetal and neonatal porcine islet tissue precludes the use of in vitro insulin secretion as a potency test as part of lot release testing; another measure of potency appropriate to fetal and neonatal cells will need to be developed for product release testing and evaluation of aliquots of these products in mouse transplant bioassays should be performed to provide meaningful post-release information.

Fat encapsulation enhances dietary nutrients utilization and growth performance of nursery pigs.

PubMed

Yang, F; Zhang, S H; Kim, S W; Ren, C X; Tian, M; Cheng, L; Song, J J; Chen, J; Chen, F; Guan, W T

2018-05-31

Encapsulation of fat may facilitate digestion and absorption of fat in nursery pigs. Two experiments were conducted to evaluate (1) effects of encapsulation of palm oil and coconut oil on growth performance, feed intake, feed efficiency, and blood parameters, and (2) effects of encapsulation of palm oil and coconut oil on apparent total tract digestibility (ATTD) of nutrients, and the activity of digestive enzymes in nursery pigs. In Exp. 1, 540 pigs (28 d of age, 8.23 ± 0.22 kg BW) were allotted to 5 treatments based on a randomized complete block design (as-fed basis). Pigs were fed basal diets with 5 different fat sources: 6.0% soybean oil (SBO), 6.0% palm oil (PO), 6.0% palm oil from encapsulated fat (EPO), 6.0% coconut oil (CO), and 6.0% coconut oil from encapsulated fat (ECO) respectively, with 6 pens per treatment and 18 pigs per pen for a 4-wk feeding trial. Dried casein and whey powder used for encapsulation were included at identical levels in all diets. Pigs fed EPO had increased (PPPad libitum for 4 weeks to measure ATTD of diets weekly and digestive enzyme activity at wk 4. Pigs fed EPO, CO, and ECO had increased (PPPEE) compared to other treatments. Pigs fed PO had greater (PP = 0.073) pancreatic lipase activity compared to other treatments whereas dietary treatments had no effect on pancreatic amylase activity. In conclusion, this study indicates that encapsulation of palm oil improved growth performance and ATTD of diets in nursery pigs, whereas the limited effects of encapsulated coconut oil were likely due to the high digestibility of the medium chain triglycerides (MCT) abundant in coconut oil.

Effects of Composition of Alginate-Polyethylene Glycol Microcapsules and Transplant Site on Encapsulated Islet Graft Outcomes in Mice

PubMed Central

Villa, Chiara; Manzoli, Vita; Abreu, Maria M.; Verheyen, Connor A.; Seskin, Michael; Najjar, Mejdi; Molano, R. Damaris; Torrente, Yvan; Ricordi, Camillo; Tomei, Alice A.

2017-01-01

Background Understanding the effects of capsule composition and transplantation site on graft outcomes of encapsulated islets will aid in the development of more effective strategies for islet transplantation without immunosuppression. Methods Here, we evaluated the effects of transplanting alginate (ALG)-based microcapsules (Micro) in the confined and well-vascularized epididymal fat pad (EFP) site, a model of the human omentum, as opposed to free-floating in the intraperitoneal cavity (IP) in mice. We also examined the effects of reinforcing ALG with polyethylene glycol (PEG). To allow transplantation in the EFP site, we minimized capsule size to 500 ± 17 μm. Unlike ALG, PEG resists osmotic stress, hence we generated hybrid microcapsules by mixing PEG and ALG (MicroMix) or by coating ALG capsules with a 15 ± 2 μm PEG layer (Double). Results We found improved engraftment of fully allogeneic BALB/c islets in Micro capsules transplanted in the EFP (median reversal time [MRT], 1 day) versus the IP site (MRT, 5 days; P < 0.01) in diabetic C57BL/6 mice and of Micro encapsulated (MRT, 8 days) versus naked (MRT, 36 days; P < 0.01) baboon islets transplanted in the EFP site. Although in vitro viability and functionality of islets within MicroMix and Double capsules were comparable to Micro, addition of PEG to ALG in MicroMix capsules improved engraftment of allogeneic islets in the IP site, but resulted deleterious in the EFP site, probably due to lower biocompatibility. Conclusions Our results suggest that capsule composition and transplant site affect graft outcomes through their effects on nutrient availability, capsule stability, and biocompatibility. PMID:27525644

Sustained glucagon-like peptide 1 expression from encapsulated transduced cells to treat obese diabetic rats.

PubMed

Moralejo, Daniel; Yanay, Ofer; Kernan, Kelly; Bailey, Adam; Lernmark, Ake; Osborne, William

2011-04-01

Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyte(TM) encapsulation devices, implanted subcutaneously and rats were monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3 ± 10.2pM that was significantly elevated over control values of 32.4 ± 2.9pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9 ± 2.3ng/ml that were significantly increased over control levels of 7.3±1.5ng/ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with α-cell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of α-cells and increased islet mass. These data suggest that encapsulated transduced cells may offer a potential long term treatment of patients. Copyright © 2010 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Sustained glucagon-like peptide 1 expression from encapsulated transduced cells to treat obese diabetic rats

PubMed Central

Moralejo, Daniel; Yanay, Ofer; Kernan, Kelly; Bailey, Adam; Lernmark, Ake; Osborne, William

2011-01-01

Obesity and type 2 diabetes (T2D) are two prevalent chronic diseases that have become a major public health concern in industrialized countries. T2D is characterized by hyperglycemia and islet beta cell dysfunction. Glucagon-like peptide 1 (GLP-1) promotes β cell proliferation and neogenesis and has a potent insulinotropic effect. Leptin receptor deficient male rats are obese and diabetic and provide a model of T2D. We hypothesized that their treatment by sustained expression of GLP-1 using encapsulated cells may prevent or delay diabetes onset. Vascular smooth muscle cells (VSMC) retrovirally transduced to secrete GLP-1 were seeded into TheraCyteTM encapsulation devices, implanted subcutaneously and rats monitored for diabetes. Rats that received cell implants showed mean plasma GLP-1 level of 119.3±10.2 pM that was significantly elevated over control values of 32.4±2.9 pM (P<0.001). GLP-1 treated rats had mean insulin levels of 45.9±2.3 ng/ml that were significantly increased over control levels of 7.3±1.5 ng/ml (P<0.001). In rats treated before diabetes onset elevations in blood glucose were delayed and rats treated after onset became normoglycemic and showed improved glucose tolerance tests. Untreated diabetic rats possess abnormal islet structures characterized by enlarged islets with β-cell infiltration and multifocal vacuolization. GLP-1 treatment induced normalization of islet structures including a mantle of β-cells and increased islet mass. These data suggest encapsulated transduced cells may offer a potential long term treatment of patients. PMID:21216666

Cryopreservation of human insulin expressing cells macro-encapsulated in a durable therapeutic immunoisolating device theracyte.

PubMed

Yakhnenko, Ilya; Wong, Wallace K; Katkov, Igor I; Itkin-Ansari, Pamela

2012-01-01

Encapsulating insulin producing cells (INPCs) in an immunoisolation device have been shown to cure diabetes in rodents without the need for immunosuppression. However, micro-encapsulation in semi-solid gels raises longevity and safety concerns for future use of stem cell derived INPCs. We have focused on a durable and retrievable macro-encapsulation (> 10(6) cells) device (TheraCyte). Cryopreservation (CP) of cells preloaded into the device is highly desirable but may require prolonged exposure to cryoprotectants during loading and post-thaw manipulations. Here, we are reporting survival and function of a human islet cell line frozen as single cells or as islet-like cell clusters. The non-clusterized cells exhibited high cryosurvival after prolonged pre-freeze or post-thaw exposure to 10 percent DMSO. However, both clusterization and especially loading INPCs into the device reduced viable yield even without CP. The survived cryopreserved macro-encapsulated INPCs remained fully functional suggesting that CP of macro-encapsulated cells is a promising tool for cell based therapies.

Mammalian Cell Encapsulation in Alginate Beads Using a Simple Stirred Vessel.

PubMed

Hoesli, Corinne A; Kiang, Roger L J; Raghuram, Kamini; Pedroza, René G; Markwick, Karen E; Colantuoni, Antonio M R; Piret, James M

2017-06-29

Cell encapsulation in alginate beads has been used for immobilized cell culture in vitro as well as for immunoisolation in vivo. Pancreatic islet encapsulation has been studied extensively as a means to increase islet survival in allogeneic or xenogeneic transplants. Alginate encapsulation is commonly achieved by nozzle extrusion and external gelation. Using this method, cell-containing alginate droplets formed at the tip of nozzles fall into a solution containing divalent cations that cause ionotropic alginate gelation as they diffuse into the droplets. The requirement for droplet formation at the nozzle tip limits the volumetric throughput and alginate concentration that can be achieved. This video describes a scalable emulsification method to encapsulate mammalian cells in 0.5% to 10% alginate with 70% to 90% cell survival. By this alternative method, alginate droplets containing cells and calcium carbonate are emulsified in mineral oil, followed by a decrease in pH leading to internal calcium release and ionotropic alginate gelation. The current method allows the production of alginate beads within 20 min of emulsification. The equipment required for the encapsulation step consists in simple stirred vessels available to most laboratories.

Construction of EMSC-islet co-localizing composites for xenogeneic porcine islet transplantation.

PubMed

Kim, Jung-Sik; Chung, Hyunwoo; Byun, Nari; Kang, Seong-Jun; Lee, Sunho; Shin, Jun-Seop; Park, Chung-Gyu

2018-03-04

Pancreatic islet transplantation is an ultimate solution for treating patients with type 1 diabetes (T1D). The pig is an ideal donor of islets for replacing scarce human islets. Besides immunological hurdles, non-immunological hurdles including fragmentation and delayed engraftment of porcine islets need solutions to succeed in porcine islet xenotransplantation. In this study, we suggest a simple but effective modality, a cell/islet co-localizing composite, to overcome these challenges. Endothelial-like mesenchymal stem cells (EMSCs), differentiated from bone-marrow derived mouse mesenchymal stem cells (MSCs), and MSCs evenly coated the surface of porcine islets (>85%) through optimized culture conditions. Both MSCs and EMSCs significantly reduced the fragmentation of porcine islets and increased the islet masses, designated as islet equivalents (IEQs). In fibrin in vitro and in vivo angiogenesis analysis, constructed EMSC-islet composites showed higher angiogenic potentials than naked islets, MSC-islet composites, or human endothelial cell-islet composites. This novel delivery method of porcine islets may have beneficial effects on the engraftment of transplanted islets by prevention of fragmentation and enhancement of revascularization. Copyright © 2018 Elsevier Inc. All rights reserved.

Current and Future Perspectives on Alginate Encapsulated Pancreatic Islet.

PubMed

Strand, Berit L; Coron, Abba E; Skjak-Braek, Gudmund

2017-04-01

Transplantation of pancreatic islets in immune protective capsules holds the promise as a functional cure for type 1 diabetes, also about 40 years after the first proof of principal study. The concept is simple in using semipermeable capsules that allow the ingress of oxygen and nutrients, but limit the access of the immune system. Encapsulated human islets have been evaluated in four small clinical trials where the procedure has been evaluated as safe, but lacking long-term efficacy. Host reactions toward the biomaterials used in the capsules may be one parameter limiting the long-term function of the graft in humans. The present article briefly discusses important capsule properties such as stability, permeability and biocompatibility, as well as possible strategies to overcome current challenges. Also, recent progress in capsule development as well as the production of insulin-producing cells from human stem cells that gives promising perspectives for the transplantation of encapsulated insulin-producing tissue is briefly discussed. Stem Cells Translational Medicine 2017;6:1053-1058. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Pancreatic Tissue Transplanted in TheraCyte Encapsulation Devices Is Protected and Prevents Hyperglycemia in a Mouse Model of Immune-Mediated Diabetes.

PubMed

Boettler, Tobias; Schneider, Darius; Cheng, Yang; Kadoya, Kuniko; Brandon, Eugene P; Martinson, Laura; von Herrath, Matthias

2016-01-01

Type 1 diabetes (T1D) is characterized by destruction of glucose-responsive insulin-producing pancreatic β-cells and exhibits immune infiltration of pancreatic islets, where CD8 lymphocytes are most prominent. Curative transplantation of pancreatic islets is seriously hampered by the persistence of autoreactive immune cells that require high doses of immunosuppressive drugs. An elegant approach to confer graft protection while obviating the need for immunosuppression is the use of encapsulation devices that allow for the transfer of oxygen and nutrients, yet prevent immune cells from making direct contact with the islet grafts. Here we demonstrate that macroencapsulation devices (TheraCyte) loaded with neonatal pancreatic tissue and transplanted into RIP-LCMV.GP mice prevented disease onset in a model of virus-induced diabetes mellitus. Histological analyses revealed that insulin-producing cells survived within the device in animal models of diabetes. Our results demonstrate that these encapsulation devices can protect from an immune-mediated attack and can contain a sufficient amount of insulin-producing cells to prevent overt hyperglycemia.

The isolation and function of porcine islets from market weight pigs.

PubMed

O'Neil, J J; Stegemann, J P; Nicholson, D T; Gagnon, K A; Solomon, B A; Mullon, C J

2001-01-01

The efficacy of clinical islet transplantation has been demonstrated with autografts, and although islet allografts have established insulin independence in a small number of IDDM patients, the treatment is confounded by the necessity of immunosuppression. the lack of donor tissue, and recurring islet immunogenicity. These limitations underscore a need to develop therapies to serve the large population of diabetic patients. Porcine islet xenotransplantation, together with a successful immune intervention strategy, may provide the necessary clinical alternative. However, a major obstacle in evaluating this approach has been the difficulty of obtaining adequate volumes of functional islet tissue from pigs. Donors of market weight are preferable to retired breeders due to their abundance, lower animal and husbandry costs. and are more suitable to meet regulatory guidelines for donor tissue for xenotransplantation. We describe a simple isolation procedure that following purification yields a mean of 350,000 IE, corresponding to 179 units of insulin and 1.8 mg of DNA with an islet purity and viability in excess of 85% (n = 317 isolations). In both short- and long-term cell cultures, porcine islets demonstrated glucose-responsive insulin secretion. However, this secretion is density dependent, which may have significant consequences in the development of immunoisolation technologies to support porcine islet xenotransplantation. Following implantation into diabetic nude mice, porcine islets remained functional in excess of 1 year. Implantation of a bioartificial pancreas containing porcine islets into pancreatectomized dogs provided significant clinical benefit with an improved diabetic condition. Finally, secretagogue-induced insulin release was demonstrated in vitro from these devices after removal from immunocompetent recipients. Immunohistochemical staining identified well-granulated islets following long-term implantation in both the rodent and canine models. This study demonstrates the ability to isolate porcine islets in clinically relevant numbers from market animals, which survive and remain functional for prolonged periods of time in an immune-deficient or immunoprotected environment.

Cryo-isolation: a novel method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue.

PubMed

Taylor, M J; Baicu, S

2011-11-01

A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment. As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods. Copyright © 2011 Elsevier Inc. All rights reserved.

Clinical application of microencapsulated islets: actual prospectives on progress and challenges.

PubMed

Calafiore, Riccardo; Basta, Giuseppe

2014-04-01

After 25 years of intense pre-clinical work on microencapsulated intraperitoneal islet grafts into non-immunosuppressed diabetic recipients, the application of this procedure to patients with type 1 diabetes mellitus has been a significant step forward. This result, achieved in a few centers worldwide, underlies the safety of biopolymers used for microencapsulation. Without this advance, no permission for human application of microcapsules would have ever been obtained after years of purification technologies applied to the raw alginates. To improve safety of the encapsulated islet graft system, renewed efforts on the capsules' bioengineering, as well as on insulin-producing cells within the capsular membranes, are in progress. It is hoped that advances in these two critical aspects of the cell encapsulation technology will result in wider human application of this system. Copyright © 2013 Elsevier B.V. All rights reserved.

Transplantation of micro- and macroencapsulated piglet islets into mice and monkeys.

PubMed

Elliott, R B; Escobar, L; Calafiore, R; Basta, G; Garkavenko, O; Vasconcellos, A; Bambra, C

2005-01-01

Neonatal porcine islets within alginate microcapsules transplanted intraperitoneally (IP) or within semi-permeable macrocapsules (TheraCyte) and transplanted subcutaneously (SC) survive and reverse diabetes for up to 16 weeks in diabetic autoimmune nonobese diabetic (NOD) mice. The islets in microcapsules transplanted IP into nondiabetic cynomolgus monkeys survived for 8 weeks. Similar results were shown with islets transplanted in TheraCytes. Neither species showed adverse effects or evidence of infection with porcine endogenous retroviruses or other endemic pig viruses. Proof of principle is illustrated for successful xenotransplantation in humans.

Pig Pancreas Anatomy: Implications for Pancreas Procurement, Preservation, and Islet Isolation

PubMed Central

Ferrer, Joana; Scott, William E; Weegman, Bradley P; Suszynski, Thomas M; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

2009-01-01

Background Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. The limited human islet supply from cadavers and poor islet yield and quality remain substantial impediments to progress in the field. Use of porcine islets holds great promise for large-scale application of islet transplantation. Consistent isolation of porcine islets is dependent on advances in pancreas procurement and preservation, and islet isolation requiring detailed knowledge of the porcine pancreatic anatomy. The primary aim of this study was to describe the vascular and ductal anatomy of the porcine pancreas in order to guide and improve organ preservation and enzyme perfusion. Methods Pancreata were removed by en bloc viscerectomy from 65 female Landrace pigs. Results 15% of organs exhibited inconsistent vascular branching from the celiac trunk. All organs had uniform patterns of branching at the superior mesenteric artery. The superior and inferior mesenteric veins (IMV) merged to become the portal vein in all but one case in which the IMV drained into the splenic vein. 97% of pancreata had three lobes: duodenal (DL), connecting (CL), and splenic (SL); 39% demonstrated ductal communication between the CL and the other two lobes; 50% had ductal communication only between the CL and DL; and 11% presented other types of ductal delineation. Conclusions Accounting for the variations in vascular and ductal anatomy, as detailed in this study, will facilitate development of protocols for preservation, optimal enzyme administration, and pancreas distention and digestion, and ultimately lead to substantial improvements in isolation outcomes. PMID:19077881

The role of genetically engineered pigs in xenotransplantation research.

PubMed

Cooper, David K C; Ekser, Burcin; Ramsoondar, Jagdeece; Phelps, Carol; Ayares, David

2016-01-01

There is a critical shortage in the number of deceased human organs that become available for the purposes of clinical transplantation. This problem might be resolved by the transplantation of organs from pigs genetically engineered to protect them from the human immune response. The pathobiological barriers to successful pig organ transplantation in primates include activation of the innate and adaptive immune systems, coagulation dysregulation and inflammation. Genetic engineering of the pig as an organ source has increased the survival of the transplanted pig heart, kidney, islet and corneal graft in non-human primates (NHPs) from minutes to months or occasionally years. Genetic engineering may also contribute to any physiological barriers that might be identified, as well as to reducing the risks of transfer of a potentially infectious micro-organism with the organ. There are now an estimated 40 or more genetic alterations that have been carried out in pigs, with some pigs expressing five or six manipulations. With the new technology now available, it will become increasingly common for a pig to express even more genetic manipulations, and these could be tested in the pig-to-NHP models to assess their efficacy and benefit. It is therefore likely that clinical trials of pig kidney, heart and islet transplantation will become feasible in the near future. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Biomimetic silica encapsultation of living cells

NASA Astrophysics Data System (ADS)

Jaroch, David Benjamin

Living cells perform complex chemical processes on size and time scales that artificial systems cannot match. Cells respond dynamically to their environment, acting as biological sensors, factories, and drug delivery devices. To facilitate the use of living systems in engineered constructs, we have developed several new approaches to create stable protective microenvironments by forming bioinspired cell-membrane-specific silica-based encapsulants. These include vapor phase deposition of silica gels, use of endogenous membrane proteins and polysaccharides as a site for silica nucleation and polycondensation in a saturated environment, and protein templated ordered silica shell formation. We demonstrate silica layer formation at the surface of pluripotent stem-like cells, bacterial biofilms, and primary murine and human pancreatic islets. Materials are characterized by AFM, SEM and EDS. Viability assays confirm cell survival, and metabolite flux measurements demonstrate normal function and no major diffusion limitations. Real time PCR mRNA analysis indicates encapsulated islets express normal levels of genetic markers for β-cells and insulin production. The silica glass encapsulant produces a secondary bone like calcium phosphate mineral layer upon exposure to media. Such bioactive materials can improve device integration with surrounding tissue upon implantation. Given the favorable insulin response, bioactivity, and long-term viability observed in silica-coated islets, we are currently testing the encapsulant's ability to prevent immune system recognition of foreign transplants for the treatment of diabetes. Such hybrid silica-cellular constructs have a wide range of industrial, environmental, and medical applications.

Minireview: Directed Differentiation and Encapsulation of Islet β-Cells—Recent Advances and Future Considerations

PubMed Central

Tse, Hubert M.; Kozlovskaya, Veronika; Kharlampieva, Eugenia

2015-01-01

Diabetes mellitus has rapidly become a 21st century epidemic with the promise to create vast economic and health burdens, if left unchecked. The 2 major forms of diabetes arise from unique causes, with outcomes being an absolute (type 1) or relative (type 2) loss of functional pancreatic islet β-cell mass. Currently, patients rely on exogenous insulin and/or other pharmacologies that restore glucose homeostasis. Although these therapies have prolonged countless lives over the decades, the striking increases in both type 1 and type 2 diabetic diagnoses worldwide suggest a need for improved treatments. To this end, islet biologists are developing cell-based therapies by which a patient's lost insulin-producing β-cell mass is replenished. Pancreatic or islet transplantation from cadaveric donors into diabetic patients has been successful, yet the functional islet demand far surpasses supply. Thus, the field has been striving toward transplantation of renewable in vitro-derived β-cells that can restore euglycemia. Challenges have been numerous, but progress over the past decade has generated much excitement. In this review we will summarize recent findings that have placed us closer than ever to β-cell replacement therapies. With the promise of cell-based diabetes therapies on the horizon, we will also provide an overview of cellular encapsulation technologies that will deliver critical protection of newly implanted cells. PMID:26340406

Effect of prolonged gelling time on the intrinsic properties of barium alginate microcapsules and its biocompatibility.

PubMed

Vaithilingam, Vijayaganapathy; Kollarikova, Gabriella; Qi, Meirigeng; Lacik, Igor; Oberholzer, Jose; Guillemin, Gilles J; Tuch, Bernard E

2011-01-01

Pericapsular fibrotic overgrowth (PFO) may be attributed to an immune response against microcapsules themselves or to antigen shedding through microcapsule pores from encapsulated islet tissue. Modification of microcapsules aimed at reducing pore size should prevent PFO and improve graft survival. This study investigated the effect of increased gelling time (20 vs. 2 min) in barium chloride on intrinsic properties of alginate microcapsules and tested their biocompatibility in vivo. Prolonged gelling time affected neither permeability nor size of the microcapsules. However, prolonged gelling time for 20 min produced brittle microcapsules compared to 2 min during compression test. Encapsulation of human islets in both types of microcapsules affected neither islet viability nor function. The presence of PFO when transplanted into a large animal model such as baboon and its absence in small animal models such as rodents suggest that the host immune response towards alginate microcapsules is species rather than alginate specific.

Quadrupole Magnetic Sorting of Porcine Islets of Langerhans

PubMed Central

Shenkman, Rustin M.; Chalmers, Jeffrey J.; Hering, Bernhard J.; Kirchhof, Nicole

2009-01-01

Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. Inconsistent isolation, purification, and recovery of large numbers of high-quality islets remain substantial impediments to progress in the field. Removing islets as soon as they are liberated from the pancreas during digestion and circumventing the need for density gradient purification is likely to result in substantially increased viable islet yields by minimizing exposure to proteolytic enzymes, reactive oxygen intermediates, and mechanical stress associated with centrifugation. This study capitalized on the hypervascularity of islets compared with acinar tissue to explore their preferential enrichment with magnetic beads to enable immediate separation in a magnetic field utilizing a quadrupole magnetic sorting. The results demonstrate that (1) preferential enrichment of porcine islets is achievable, but homogeneous bead distribution within the pancreas is difficult to achieve with current protocols; (2) greater than 70% of islets in the dissociated pancreatic tissue were recovered by quadrupole magnetic sorting, but their purity was low; and (3) infused islets purified by density gradients and subsequently passed through quadrupole magnetic sorting had similar potency as uninfused islets. These results demonstrate proof of concept and define the steps for implementation of this technology in pig and human islet isolation. PMID:19505179

Frankenswine, or bringing home the bacon

PubMed Central

2008-01-01

Xenotransplantation—specifically from pig into human—could resolve the critical shortage of organs, tissues and cells for clinical transplantation. Genetic engineering techniques in pigs are relatively well-developed and to date have largely been aimed at producing pigs that either (1) express high levels of one or more human complement-regulatory protein(s), such as decay-accelerating factor or membrane cofactor protein, or (2) have deletion of the gene responsible for the expression of the oligosaccharide, Galα1,3Gal (Gal), the major target for human anti-pig antibodies, or (3) have both manipulations. Currently the transplantation of pig organs in adequately-immunosuppressed baboons results in graft function for periods of 2–6 months (auxiliary hearts) and 2–3 months (life-supporting kidneys). Pig islets have maintained normoglycemia in diabetic monkeys for >6 months. The remaining immunologic barriers to successful xenotransplantation are discussed, and brief reviews made of (1) the potential risk of the transmission of an infectious microorganism from pig to patient and possibly to the public at large, (2) the potential physiologic incompatibilities between a pig organ and its human counterpart, (3) the major ethical considerations of clinical xenotransplantation, and (4) the possible alternatives that compete with xenotransplantation in the field of organ or cell replacement, such as mechanical devices, tissue engineering, stem cell biology and organogenesis. Finally, the proximity of clinical trials is discussed. Islet xenotransplantation is already at the stage where clinical trials are actively being considered, but the transplantation of pig organs will probably require further genetic modifications to be made to the organ-source pigs to protect their tissues from the coagulation/anticoagulation dysfunction that plays a significant role in pig graft failure after transplantation in primates. PMID:19279708

Progress and challenges of the bioartificial pancreas

NASA Astrophysics Data System (ADS)

Hwang, Patrick T. J.; Shah, Dishant K.; Garcia, Jacob A.; Bae, Chae Yun; Lim, Dong-Jin; Huiszoon, Ryan C.; Alexander, Grant C.; Jun, Ho-Wook

2016-11-01

Pancreatic islet transplantation has been validated as a treatment for type 1 diabetes since it maintains consistent and sustained type 1 diabetes reversal. However, one of the major challenges in pancreatic islet transplantation is the body's natural immune response to the implanted islets. Immunosuppressive drug treatment is the most popular immunomodulatory approach for islet graft survival. However, administration of immunosuppressive drugs gives rise to negative side effects, and long-term effects are not clearly understood. A bioartificial pancreas is a therapeutic approach to enable pancreatic islet transplantation without or with minimal immune suppression. The bioartificial pancreas encapsulates the pancreatic islets in a semi-permeable environment which protects islets from the body's immune responses, while allowing the permeation of insulin, oxygen, nutrients, and waste. Many groups have developed various types of the bioartificial pancreas and tested their efficacy in animal models. However, the clinical application of the bioartificial pancreas still requires further investigation. In this review, we discuss several types of bioartificial pancreases and address their advantages and limitations. We also discuss recent advances in bioartificial pancreas applications with microfluidic or micropatterning technology.

Bioorthogonal layer-by-layer encapsulation of pancreatic islets via hyperbranched polymers

PubMed Central

Gattás-Asfura, Kerim M.; Stabler, Cherie L.

2013-01-01

The encapsulation of viable tissues via layer-by-layer polymer assembly provides a versatile platform for cell surface engineering, with nanoscale control over capsule properties. Herein, we report the development of a hyperbranched polymer-based, ultrathin capsule architecture expressing bioorthogonal functionality and tailored physiochemical properties. Random carbodiimide-based condensation of 3,5-dicarboxyphenyl glycineamide on alginate yielded a highly branched polysaccharide with multiple, spatially restricted, and readily functionalizable terminal carboxylate moieties. Poly(ethylene glycol) (PEG) was utilized to link azido end groups to the structured alginate. Together with phosphine functionalized poly(amido amine) (PAMAM) dendrimer, nanoscale layer-by-layer coatings, covalently stabilized via Staudinger ligation, were assembled onto solid surfaces and pancreatic islets. The effects of electrostatic and/or bioorthogonal covalent interlayer interactions on the resulting coating efficiency and stability, as well as pancreatic islet viability and function, were studied. These hyperbranched polymers provide a flexible platform for the formation of covalently stabilized ultrathin coatings on viable cells and tissues. In addition, the hyperbranched nature of the polymers presents a highly functionalized surface capable of bioorthogonal conjugation of additional bioactive or labeling motifs. PMID:24063764

Durable Control of Autoimmune Diabetes in Mice Achieved by Intraperitoneal Transplantation of “Neo‐Islets,” Three‐Dimensional Aggregates of Allogeneic Islet and “Mesenchymal Stem Cells”

PubMed Central

Gooch, Anna; Hu, Zhuma; Ahlstrom, Jon; Zhang, Ping

2017-01-01

Abstract Novel interventions that reestablish endogenous insulin secretion and thereby halt progressive end‐organ damage and prolong survival of patients with autoimmune Type 1 diabetes mellitus (T1DM) are urgently needed. While this is currently accomplished with allogeneic pancreas or islet transplants, their utility is significantly limited by both the scarcity of organ donors and life‐long need for often‐toxic antirejection drugs. Coadministering islets with bone marrow‐derived mesenchymal stem cells (MSCs) that exert robust immune‐modulating, anti‐inflammatory, anti‐apoptotic, and angiogenic actions, improves intrahepatic islet survival and function. Encapsulation of insulin‐producing cells to prevent immune destruction has shown both promise and failures. Recently, stem cell‐derived insulin secreting β‐like cells induced euglycemia in diabetic animals, although their clinical use would still require encapsulation or anti‐rejection drugs. Instead of focusing on further improvements in islet transplantation, we demonstrate here that the intraperitoneal administration of islet‐sized “Neo‐Islets” (NIs), generated by in vitro coaggregation of allogeneic, culture‐expanded islet cells with high numbers of immuno‐protective and cyto‐protective MSCs, resulted in their omental engraftment in immune‐competent, spontaneously diabetic nonobese diabetic (NOD) mice. This achieved long‐term glycemic control without immunosuppression and without hypoglycemia. In preparation for an Food and Drug Administration‐approved clinical trial in dogs with T1DM, we show that treatment of streptozotocin‐diabetic NOD/severe combined immunodeficiency mice with identically formed canine NIs produced durable euglycemia, exclusively mediated by dog‐specific insulin. We conclude that this novel technology has significant translational relevance for canine and potentially clinical T1DM as it effectively addresses both the organ donor scarcity (>80 therapeutic NI doses/donor pancreas can be generated) and completely eliminates the need for immunosuppression. Stem Cells Translational Medicine 2017;6:1631–1643 PMID:28467694

Microfabricated biocapsules for the immunoisolation of pancreatic islets of Langerhans

NASA Astrophysics Data System (ADS)

Desai, Tejal Ashwin

1998-08-01

A silicon-based microfabricated biocapsule was developed and evaluated for use in the immunoisolation of transplanted cells, specifically pancreatic islets of Langerhans for the treatment of Type I diabetes. The transplantation of cells with specific functions is a promising therapy for a wide variety of pathologies including diabetes, Parkinson's, and hemophilia. Such transplanted cells, however, are sensitive to both cellular and humoral immune rejection as well as damage by autoimmune activity, without chronic immunosuppression. The research presented in this dissertation investigated whether microfabricated silicon-based biocapsules, with uniform membrane pore sizes in the tens of nanometer range, could provide an immunoprotective environment for pancreatic islets and other insulin-secreting cell lines, while maintaining cell viability and functionality. By utilizing fabrication techniques commonly employed in the microelectronics industry (MEMS), membranes were fabricated with precisely controlled and uniform pore sizes, allowing the optimization of biocapsule membrane parameters for the encapsulation of specific hormone-secreting cell types. The biocapsule-forming process employed bulk micromachining to define cell-containing chambers within single crystalline silicon wafers. These chambers interface with the surrounding biological environment through polycrystalline silicon filter membranes, which were surface micromachined to present a high density of uniform pores to allow sufficient permeability to oxygen, glucose, and insulin. Both in vitro and in vivo experiments established the biocompatibility of the microfabricated biocapsule, and demonstrated that encapsulated cells could live and function normally in terms of insulin-secretion within microfabricated environments for extended periods of time. This novel research shows the potential of using microfabricated biocapsules for the encapsulation of several different cell xenografts. The semipermeability of microfabricated biocapsules, their biocompatibility, along with their thermal and chemical stability, may provide an improved encapsulation device for the immunoisolation of cell xenografts in hormone-replacement and cell-based therapies.

[Islet transplantation as a treatment for complications of type I diabetes].

PubMed

Wszoła, Michał; Kwiatkowski, Artur; Berman, Andrzej; Górski, Łukasz; Chmura, Andrzej

2013-09-01

Reduced physical activity and high calories up-take along with carbohydrates based diet are considered to be a leading cause of diabetes mellitus rise in western countries. Together with rise in DM morbidity, increase of complicated diabetes is also observed. Pancreas transplantation occurred to be a milestone in diabetic patient management. Guine pig pancreatic islets isolation performed for the first time by Moskalewski in 1965 and updates of his method have given an opportunity to introduce allogenic isolated islets transplantation to clinical usage. For the first time in Poland clinical allotransplantation of isolated pancreatic islets took place in Department of General Surgery and Transplantology of Medical University of Warsaw in 12's June 2008. Unfortunately, unsatisfying results of islet transplantation, specially short period of insulin independence after successful transplantation related with multifactor islet function lost, reduce clinical indications. In this publication we have analyzed known and potential factors of islet lost and we have tried to find way to prevent them, with a long period insulin-independence after transplantation as a main goal.

Biomolecular strategies for cell surface engineering

NASA Astrophysics Data System (ADS)

Wilson, John Tanner

Islet transplantation has emerged as a promising cell-based therapy for the treatment of diabetes, but its clinical efficacy remains limited by deleterious host responses that underlie islet destruction. In this dissertation, we describe the assembly of ultrathin conformal coatings that confer molecular-level control over the composition and biophysicochemical properties of the islet surface with implications for improving islet engraftment. Significantly, this work provides novel biomolecular strategies for cell surface engineering with broad biomedical and biotechnological applications in cell-based therapeutics and beyond. Encapsulation of cells and tissue offers a rational approach for attenuating deleterious host responses towards transplanted cells, but a need exists to develop cell encapsulation strategies that minimize transplant volume. Towards this end, we endeavored to generate nanothin films of diverse architecture with tunable properties on the extracellular surface of individual pancreatic islets through a process of layer-by-layer (LbL) self assembly. We first describe the formation of poly(ethylene glycol) (PEG)-rich conformal coatings on islets via LbL self assembly of poly(L-lysine)-g-PEG(biotin) and streptavidin. Multilayer thin films conformed to the geometrically and chemically heterogeneous islet surface, and could be assembled without loss of islet viability or function. Significantly, coated islets performed comparably to untreated controls in a murine model of allogenic intraportal islet transplantation, and, to our knowledge, this is the first study to report in vivo survival and function of nanoencapsulated cells or cell aggregates. Based on these findings, we next postulated that structurally similar PLL-g-PEG copolymers comprised of shorter PEG grafts might be used to initiate and propagate the assembly of polyelectrolyte multilayer (PEM) films on pancreatic islets, while simultaneously preserving islet viability. Through control of PLL backbone molecular weight, PEG chain length, and grafting ratio, PLL-g-PEG copolymers were rendered cytocompatible and used to initiate and propagate the growth of cell surface-supported PEM films. Planar characterization of this novel class of PEM films indicated that film thickness and composition may be tailored through appropriate control of layer number and copolymer properties. Furthermore, these investigations have helped establish a conceptual framework for the rational design of cell surface-supported thin films, with the objective of translating the diverse biomedical and biotechnological applications of PEM films to cellular interfaces. Important to the development of effective conformal islet coatings is an inherent strategy through which to incorporate bioactive molecules for directing desired biochemical or cellular responses. Towards this end, PLL-g-PEG copolymers functionalized with biotin, azide, and hydrazide moieties were synthesized and used, either alone or in combination, to capture streptavidin-, triphenylphosphine-, and aldehyde-labeled probes, respectively, on the islet surface. Additionally, PEM films assembled using alginate chemically modified to contain aldehyde groups could be used to introduce hydrazide-functionalized molecules to the islet surface. Hence, modified film constituents may be used as modular elements for controlling the chemical composition cell and tissue surfaces. Finally, we report a strategy for tethering thrombomodulin (TM) to the islet surface. Through site-specific, C-terminal biotinylation of TM and optimization of cell surface biotinylation, TM could be integrated with the islet surface. Re-engineering of islet surfaces with TM resulted in an increased catalytic capacity of islets to generate the powerful anti-inflammatory agent, activated protein C (APC), thereby providing a facile strategy for increasing the local concentration of APC at the site of transplantation.

Enhancing human islet transplantation by localized release of trophic factors from PLG scaffolds.

PubMed

Hlavaty, K A; Gibly, R F; Zhang, X; Rives, C B; Graham, J G; Lowe, W L; Luo, X; Shea, L D

2014-07-01

Islet transplantation represents a potential cure for type 1 diabetes, yet the clinical approach of intrahepatic delivery is limited by the microenvironment. Microporous scaffolds enable extrahepatic transplantation, and the microenvironment can be designed to enhance islet engraftment and function. We investigated localized trophic factor delivery in a xenogeneic human islet to mouse model of islet transplantation. Double emulsion microspheres containing exendin-4 (Ex4) or insulin-like growth factor-1 (IGF-1) were incorporated into a layered scaffold design consisting of porous outer layers for islet transplantation and a center layer for sustained factor release. Protein encapsulation and release were dependent on both the polymer concentration and the identity of the protein. Proteins retained bioactivity upon release from scaffolds in vitro. A minimal human islet mass transplanted on Ex4-releasing scaffolds demonstrated significant improvement and prolongation of graft function relative to blank scaffolds carrying no protein, and the release profile significantly impacted the duration over which the graft functioned. Ex4-releasing scaffolds enabled better glycemic control in animals subjected to an intraperitoneal glucose tolerance test. Scaffolds releasing IGF-1 lowered blood glucose levels, yet the reduction was insufficient to achieve euglycemia. Ex4-delivering scaffolds provide an extrahepatic transplantation site for modulating the islet microenvironment to enhance islet function posttransplant. © Copyright 2014 The American Society of Transplantation and the American Society of Transplant Surgeons.

A new scaffold containing small intestinal submucosa and mesenchymal stem cells improves pancreatic islet function and survival in vitro and in vivo

PubMed Central

Wang, Dan; Ding, Xiaoming; Xue, Wujun; Zheng, Jin; Tian, Xiaohui; Li, Yang; Wang, Xiaohong; Song, Huanjin; Liu, Hua; Luo, Xiaohui

2017-01-01

It is unknown whether a scaffold containing both small intestinal submucosa (SIS) and mesenchymal stem cells (MSCs) for transplantation may improve pancreatic islet function and survival. In this study, we examined the effects of a SIS-MSC scaffold on islet function and survival in vitro and in vivo. MSCs and pancreatic islets were isolated from Sprague-Dawley rats, and SIS was isolated from Bamei pigs. The islets were apportioned among 3 experimental groups as follows: SIS-islets, SIS-MSC-islets and control-islets. In vitro, islet function was measured by a glucose-stimulated insulin secretion test; cytokines in cultured supernatants were assessed by enzyme-linked immunosorbent assay; and gene expression was analyzed by reverse transcription-quantitative PCR. In vivo, islet transplantation was performed in rats, and graft function and survival were monitored by measuring the blood glucose levels. In vitro, the SIS-MSC scaffold was associated with improved islet viability and enhanced insulin secretion compared with the controls, as well as with the increased the expression of insulin 1 (Ins1), pancreatic and duodenal homeobox 1 (Pdx1), platelet endothelial cell adhesion molecule 1 [Pecam1; also known as cluster of differentiation 31 (CD31)] and vascular endothelial growth factor A (Vegfa) in the islets, increased growth factor secretion, and decreased tumor necrosis factor (TNF) secretion. In vivo, the SIS-MSC scaffold was associated with improved islet function and graft survival compared with the SIS and control groups. On the whole, our findings demonstrate that the SIS-MSC scaffold significantly improved pancreatic islet function and survival in vitro and in vivo. This improvement may be associated with the upregulation of insulin expression, the improvement of islet microcirculation and the secretion of cytokines. PMID:27909715

Anti-inflammatory thalidomide improves islet grafts survival and functions in a xenogenic environment.

PubMed

Chen, Chunguang; Kuehn, Carina; Bretzel, Reinhard G; Linn, Thomas

2009-07-20

Thalidomide possesses both anti-inflammatory and anti-angiogenic properties. This study investigates its potential application in islet transplantation with a xenogenic transplantation model. Transplantation was performed using C57Bl/6 mice and NMRI nu/nu mice as recipients of porcine islets. Moreover, islet graft vasculature and inflammation were investigated to identify the mechanisms of thalidomide action. In the immunocompetent environment of C57Bl/6 mice, a fast graft rejection was observed. The group treated with thalidomide 200 mg/kg BW per day achieved and maintained euglycemia in the complete observation period for 42 days. The treated mice had more functional islet graft mass with less leukocyte infiltration. The pro-inflammatory TNF-alpha and VEGF content in islet grafted kidneys was significantly lowered by the treatment. By comparison, thalidomide was not effective in improving graft survival in immunocompromised nude mice. It strongly inhibited the VEGF and TNF-alpha-induced endothelial proliferation of isolated pig islets in a dose dependent manner. The magnitude of thalidomide's inhibitory effect was nearly identical to the effect of VEGF- receptor 2 inhibitor SU416 and anti-TNF-receptor 1 neutralizing antibody, and was reversed by sphingosine-1-phosphate. In conclusion, the anti-inflammatory effect of thalidomide improved islet graft survival and function in a transplantation model with a maximum immune barrier.

Improved survival of macroencapsulated islets of Langerhans by preimplantation of the immunoisolating device: a morphometric study.

PubMed

Rafael, E; Wu, G S; Hultenby, K; Tibell, A; Wernerson, A

2003-01-01

Encapsulation of cells in a semipermeable membrane may in the future provide an opportunity to treat a variety of endocrine and neurological disorders, without the need for lifelong immunosuppression. The physiological conditions in the device are crucial factors for graft survival. Previously, we have shown that the exchange across the immunoisolating membrane and the microcirculation around the TheraCyte device increase around 3 months after implantation. The aim of this study was to determine whether preimplantation of the TheraCyte device would improve the survival of a later transplanted islet graft. A TheraCyte device was implanted SC on one side of the back of a nondiabetic SD rat. After 3 months, 1500 islets isolated from SD rats were transplanted via the device port. At the same time, another device, loaded with the same number of islets, was implanted on the other side of the back. Both devices were explanted 2 weeks after islet transplantation (i.e., 3.5 months and 0.5 month after device implantation, respectively). Six pairs of devices were evaluated by morphometery. The volume densities of viable islets were 0.22 +/- 0.04 in the preimplanted device vs. 0.06 +/- 0.03 in the nonpreimplanted one (p < 0.05). The corresponding volume densities of fibrosis and necrosis were 0.64 +/- 0.13 vs. 0.85 +/- 0.08 (p < 0.05) and 0.11 +/- 0.14 vs. 0.09 +/- 0.07 (ns), respectively. When the absolute volumes (mm3) were calculated, preimplanted devices contained 1.1 +/- 0.7 endocrine cells while nonpreimplanted ones contained 0.4 +/- 0.2 (p < 0.05). The percentages of insulin- positive beta-cells in the preimplanted versus nonpreimplanted device were 80 +/- 5% and 67 +/- 6%, respectively (p < 0.01). The corresponding volumes of fibrotic tissue were 3.0 +/- 1.8 vs. 5.2 +/- 1.2 (p < 0.05), while the amount of necrotic tissue did not differ significantly (0.42 +/- 0.5 vs. 0.50 +/- 0.3). Preimplantation of the TheraCyte device seems to improve the survival of an encapsulated islet graft and reduce fibroblast outgrowth in the device.

Production and characterization of alginate microcapsules produced by a vibrational encapsulation device.

PubMed

Mazzitelli, S; Tosi, A; Balestra, C; Nastruzzi, C; Luca, G; Mancuso, F; Calafiore, R; Calvitti, M

2008-09-01

The optimization, through a Design of Experiments (DoE) approach, of a microencapsulation procedure for isolated neonatal porcine islets (NPI) is described. The applied method is based on the generation of monodisperse droplets by a vibrational nozzle. An alginate/polyornithine encapsulation procedure, developed and validated in our laboratory for almost a decade, was used to embody pancreatic islets. We analyzed different experimental parameters including frequency of vibration, amplitude of vibration, polymer pumping rate, and distance between the nozzle and the gelling bath. We produced calcium-alginate gel microbeads with excellent morphological characteristics as well as a very narrow size distribution. The automatically produced microcapsules did not alter morphology, viability and functional properties of the enveloped NPI. The optimization of this automatic procedure may provide a novel approach to obtain a large number of batches possibly suitable for large scale production of immunoisolated NPI for in vivo cell transplantation procedures in humans.

Degradation of phosphor-in-glass encapsulants with various phosphor types for high power LEDs

NASA Astrophysics Data System (ADS)

Iqbal, Fauzia; Kim, Sunil; Kim, Hyungsun

2017-10-01

In order to replace conventional silicone-based phosphor light emitting diodes (LEDs), inorganic color converters with high thermal stabilities and transparencies, i.e., phosphors-in-glass (PiGs), have been investigated as encapsulants for high-power LEDs. In this paper, the effect of various types of phosphors, i.e., LuAG (green, Lu3Al5O12:Ce3+), silicate (yellow, Sr2SiO4:Eu2+), CASN (red, CaAlSiN3:Eu2+), and oxynitride (yellow, (Sr,Ba) Si2O2N2:Eu2+), on the reliability/degradation of the remote PiG encapsulants is explored for high power LEDs. For this purpose, a glass composition (SiO2-B2O3-ZnO-Na2O) was separately mixed with each type of phosphor and then sintered at appropriate temperatures to make the corresponding PiG. The reliabilities of the formed PiGs were evaluated by standard accelerated-aging tests (85 °C/85% RH) for 1000 h. Luminosity losses and shifts in the Commission Internationale de l'Eclairage (CIE) coordinates of the PiGs were measured before and after aging. Thermal, and moisture-induced quenching behavior was also analyzed. The surface of PiGs with different phosphors degraded differently, possibly because of structural incompatibilities between the glass matrix and phosphor type. Determining the compatibility of the glass composition with the type of phosphor used is therefore important in order to ensure the long-term stabilities of encapsulants for use in commercial LEDs.

Microencapsulation of pancreatic islets with canine ear cartilage for immunoisolation.

PubMed

Lee, J I; Kim, H W; Kim, J Y; Bae, S J; Joo, D J; Huh, K H; Fang, Y H; Jeong, J H; Kim, M S; Kim, Y S

2012-05-01

Improving human islet transplantation is often limited by the shortage of donors and the side effects of immunosuppressive agents. If immunoisolation is properly used, it can overcome these obstacles. Because artificial materials are adopted in this technique, however, there are still multiple issues with biocompatibility and foreign body reactions. We developed a chondrocyte microencapsulated immunoisolated islet (CMI-islet) that allows living cells to act as the immunoisolating material. To manufacture CMI-islets for xenotransplantation, isolated rat pancreatic islets were placed on low cell-binding culture dishes. Subsequently, expanded canine auricular cartiage primary cells were seeded on these dishes at a high density and maintained in a suspended state via a shaking culture system. Morphological evaluations showed good islet viability and a clear progression of the islet- encapsulation events. When the cells were challenged with glucose, they were able to secrete sufficient insulin according to glucose concentrations. The CMI-islets responded better to the glucose challenge than did nude pancreatic islets and created better glucose-insulin feedback regulation. Moreover, insulin secretion into the culture medium was confirmed over a period of 100 days, showing the survival and secretory capacity of the CMI-islet cells. By microencapsulating pancreatic islets with recipient ear cartilage cells, long-term insulin secretion can be maintained and the response to glucose challenges improved. This new immunodelusion technology differs from other immunoisolation techniques in that the donor tissue is enclosed with the recipient's tissue, thus allowing the transplanted cells to be recognized as recipient cells. This microencapsulation method may lead to developing viable xenotransplantation techniques that do not use immunosuppressive drugs. Copyright © 2012 Elsevier Inc. All rights reserved.

Membranes to achieve immunoprotection of transplanted islets

PubMed Central

Schweicher, Julien; Nyitray, Crystal; Desai, Tejal A.

2014-01-01

Transplantation of islet or beta cells is seen as the cure for type 1 diabetes since it allows physiological regulation of blood glucose levels without requiring any compliance from the patients. In order to circumvent the use of immunosuppressive drugs (and their side effects), semipermeable membranes have been developed to encapsulate and immunoprotect transplanted cells. This review presents the historical developments of immunoisolation and provides an update on the current research in this field. A particular emphasis is laid on the fabrication, characterization and performance of membranes developed for immunoisolation applications. PMID:24389172

Pancreatic cell immobilization in alginate beads produced by emulsion and internal gelation.

PubMed

Hoesli, Corinne A; Raghuram, Kamini; Kiang, Roger L J; Mocinecová, Dušana; Hu, Xiaoke; Johnson, James D; Lacík, Igor; Kieffer, Timothy J; Piret, James M

2011-02-01

Alginate has been used to protect transplanted pancreatic islets from immune rejection and as a matrix to increase the insulin content of islet progenitor cells. The throughput of alginate bead generation by the standard extrusion and external gelation method is limited by the rate of droplet formation from nozzles. Alginate bead generation by emulsion and internal gelation is a scaleable alternative that has been used with biological molecules and microbial cells, but not mammalian cells. We describe the novel adaptation of this process to mammalian cell immobilization. After optimization, the emulsion process yielded 90 ± 2% mouse insulinoma 6 (MIN6) cell survival, similar to the extrusion process. The MIN6 cells expanded at the same rate in both bead types to form pseudo-islets with increased glucose stimulation index compared to cells in suspension. The emulsion process was suitable for primary pancreatic exocrine cell immobilization, leading to 67 ± 32 fold increased insulin expression after 10 days of immobilized culture. Due to the scaleability and broad availability of stirred mixers, the emulsion process represents an attractive option for laboratories that are not equipped with extrusion-based cell encapsulators, as well as for the production of immobilized or encapsulated cellular therapeutics on a clinical scale. © 2010 Wiley Periodicals, Inc.

Anti-Inflammatory Thalidomide Improves Islet Grafts Survival and Functions in a Xenogenic Environment

PubMed Central

Chen, Chunguang; Kuehn, Carina; Bretzel, Reinhard G.; Linn, Thomas

2009-01-01

Thalidomide possesses both anti-inflammatory and anti-angiogenic properties. This study investigates its potential application in islet transplantation with a xenogenic transplantation model. Transplantation was performed using C57Bl/6 mice and NMRI nu/nu mice as recipients of porcine islets. Moreover, islet graft vasculature and inflammation were investigated to identify the mechanisms of thalidomide action. In the immunocompetent environment of C57Bl/6 mice, a fast graft rejection was observed. The group treated with thalidomide 200 mg/kg BW per day achieved and maintained euglycemia in the complete observation period for 42 days. The treated mice had more functional islet graft mass with less leukocyte infiltration. The pro-inflammatory TNF-α and VEGF content in islet grafted kidneys was significantly lowered by the treatment. By comparison, thalidomide was not effective in improving graft survival in immunocompromised nude mice. It strongly inhibited the VEGF and TNF-α-induced endothelial proliferation of isolated pig islets in a dose dependent manner. The magnitude of thalidomide's inhibitory effect was nearly identical to the effect of VEGF- receptor 2 inhibitor SU416 and anti-TNF-receptor 1 neutralizing antibody, and was reversed by sphingosine-1-phosphate. In conclusion, the anti-inflammatory effect of thalidomide improved islet graft survival and function in a transplantation model with a maximum immune barrier. PMID:19617916

Effect of hyperglycaemia on muscarinic M3 receptor expression and secretory sensitivity to cholinergic receptor activation in islets.

PubMed

Hauge-Evans, A C; Reers, C; Kerby, A; Franklin, Z; Amisten, S; King, A J; Hassan, Z; Vilches-Flores, A; Tippu, Z; Persaud, S J; Jones, P M

2014-10-01

Islets are innervated by parasympathetic nerves which release acetylcholine (ACh) to amplify glucose-induced insulin secretion, primarily via muscarinic M3 receptors (M3R). Here we investigate the consequence of chronic hyperglycaemia on islet M3R expression and secretory sensitivity of mouse islets to cholinergic receptor activation. The impact of hyperglycaemia was studied in (i) islets isolated from ob/ob mice, (ii) alginate-encapsulated mouse islets transplanted intraperitoneally into streptozotocin-induced diabetic mice and (iii) mouse and human islets maintained in vitro at 5.5 or 16 mmol/l glucose. Blood glucose levels were assessed by a commercial glucose meter, insulin content by RIA and M3R expression by qPCR and immunohistochemistry. M3R mRNA expression was reduced in both ob/ob islets and islets maintained at 16 mmol/l glucose for 3 days (68 and 50% control, respectively). In all three models of hyperglycaemia the secretory sensitivity to the cholinergic receptor agonist, carbachol, was reduced by 60-70% compared to control islets. Treatment for 72 h with the irreversible PKC activator, PMA, or the PKC inhibitor, Gö6983, did not alter islet M3R mRNA expression nor did incubation with the PI3K-inhibitor, LY294002, although enhancement of glucose-induced insulin secretion by LY294002 was reduced in islets maintained at 16 mmol/l glucose, as was mRNA expression of the PI3K regulatory subunit, p85α. Cholinergic regulation of insulin release is impaired in three experimental islet models of hyperglycaemia consistent with reduced expression of M3 receptors. Our data suggest that the receptor downregulation is a PKC- and PI3K-independent consequence of the hyperglycaemic environment, and they imply that M3 receptors could be potential targets in the treatment of type 2 diabetes. © 2014 John Wiley & Sons Ltd.

Design, characterisation and application of alginate-based encapsulated pig liver esterase.

PubMed

Pauly, Jan; Gröger, Harald; Patel, Anant V

2018-06-05

Encapsulation of hydrolases in biopolymer-based hydrogels often suffers from low activities and encapsulation efficiencies along with high leaching and unsatisfactory recycling properties. Exemplified for the encapsulation of pig liver esterase the coating of alginate and chitosan beads have been studied by creating various biopolymer hydrogel beads. Enzyme activity and encapsulation efficiency were notably enhanced by chitosan coating of alginate beads while leaching remained nearly unchanged. This was caused by the enzymatic reaction acidifying the matrix, which increased enzyme retention through enhanced electrostatic enzyme-alginate interaction but decreased activity through enzyme deactivation. A practical and ready-to-use method for visualising pH in beads during reaction by co-encapsulation of a conventional pH indicator was also found. Our method proves that pH control inside the beads can only be realised by buffering. The resulting beads provided a specific activity of 0.267 μmol ∙ min -1 ∙ mg -1 , effectiveness factor 0.88, encapsulation efficiency of 88%, 5% leaching and good recycling properties. This work will contribute towards better understanding and application of encapsulated hydrolases for enzymatic syntheses. Copyright © 2018 Elsevier B.V. All rights reserved.

Size- and shape-dependent foreign body immune response to materials implanted in rodents and non-human primates

NASA Astrophysics Data System (ADS)

Veiseh, Omid; Doloff, Joshua C.; Ma, Minglin; Vegas, Arturo J.; Tam, Hok Hei; Bader, Andrew R.; Li, Jie; Langan, Erin; Wyckoff, Jeffrey; Loo, Whitney S.; Jhunjhunwala, Siddharth; Chiu, Alan; Siebert, Sean; Tang, Katherine; Hollister-Lock, Jennifer; Aresta-Dasilva, Stephanie; Bochenek, Matthew; Mendoza-Elias, Joshua; Wang, Yong; Qi, Merigeng; Lavin, Danya M.; Chen, Michael; Dholakia, Nimit; Thakrar, Raj; Lacík, Igor; Weir, Gordon C.; Oberholzer, Jose; Greiner, Dale L.; Langer, Robert; Anderson, Daniel G.

2015-06-01

The efficacy of implanted biomedical devices is often compromised by host recognition and subsequent foreign body responses. Here, we demonstrate the role of the geometry of implanted materials on their biocompatibility in vivo. In rodent and non-human primate animal models, implanted spheres 1.5 mm and above in diameter across a broad spectrum of materials, including hydrogels, ceramics, metals and plastics, significantly abrogated foreign body reactions and fibrosis when compared with smaller spheres. We also show that for encapsulated rat pancreatic islet cells transplanted into streptozotocin-treated diabetic C57BL/6 mice, islets prepared in 1.5-mm alginate capsules were able to restore blood-glucose control for up to 180 days, a period more than five times longer than for transplanted grafts encapsulated within conventionally sized 0.5-mm alginate capsules. Our findings suggest that the in vivo biocompatibility of biomedical devices can be significantly improved simply by tuning their spherical dimensions.

Stem Cell Therapies for Treating Diabetes: Progress and Remaining Challenges.

PubMed

Sneddon, Julie B; Tang, Qizhi; Stock, Peter; Bluestone, Jeffrey A; Roy, Shuvo; Desai, Tejal; Hebrok, Matthias

2018-06-01

Restoration of insulin independence and normoglycemia has been the overarching goal in diabetes research and therapy. While whole-organ and islet transplantation have become gold-standard procedures in achieving glucose control in diabetic patients, the profound lack of suitable donor tissues severely hampers the broad application of these therapies. Here, we describe current efforts aimed at generating a sustainable source of functional human stem cell-derived insulin-producing islet cells for cell transplantation and present state-of-the-art efforts to protect such cells via immune modulation and encapsulation strategies. Copyright © 2018. Published by Elsevier Inc.

Noninvasive evaluation of the vascular response to transplantation of alginate encapsulated islets using the dorsal skin-fold model.

PubMed

Krishnan, Rahul; Arora, Rajan P; Alexander, Michael; White, Sean M; Lamb, Morgan W; Foster, Clarence E; Choi, Bernard; Lakey, Jonathan R T

2014-01-01

Alginate encapsulation reduces the risk of transplant rejection by evading immune-mediated cell injury and rejection; however, poor vascular perfusion results in graft failure. Since existing imaging models are incapable of quantifying the vascular response to biomaterial implants after transplantation, in this study, we demonstrate the use of in vivo laser speckle imaging (LSI) and wide-field functional imaging (WiFI) to monitor the microvascular environment surrounding biomaterial implants. The vascular response to two islet-containing biomaterial encapsulation devices, alginate microcapsules and a high-guluronate alginate sheet, was studied and compared after implantation into the mouse dorsal window chamber (N = 4 per implant group). Images obtained over a 14-day period using LSI and WiFI were analyzed using algorithms to quantify blood flow, hemoglobin oxygen saturation and vascular density. Using our method, we were able to monitor the changes in the peri-implant microvasculature noninvasively without the use of fluorescent dyes. Significant changes in blood flow, hemoglobin oxygen saturation and vascular density were noted as early as the first week post-transplant. The dorsal window chamber model enables comparison of host responses to transplanted biomaterials. Future experiments will study the effect of changes in alginate composition on the vascular and immune responses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Islet Transplantation in Type 1 Diabetes: Ongoing Challenges, Refined Procedures, and Long-Term Outcome

PubMed Central

Shapiro, A.M. James

2012-01-01

Remarkable progress has been made in islet transplantation over a span of 40 years. Once just an experimental curiosity in mice, this therapy has moved forward, and can now provide robust therapy for highly selected patients with type 1 diabetes (T1D), refractory to stabilization by other means. This progress could not have occurred without extensive dynamic international collaboration. Currently, 1,085 patients have undergone islet transplantation at 40 international sites since the Edmonton Protocol was reported in 2000 (752 allografts, 333 autografts), according to the Collaborative Islet Transplant Registry. The long-term results of islet transplantation in selected centers now match registry data of pancreas-alone transplantation, with 6 sites reporting five-year insulin independence rates ≥50%. Islet transplantation has been criticized for the use of multiple donor pancreas organs, but progress has also occurred in single-donor success, with 10 sites reporting increased single-donor engraftment. The next wave of innovative clinical trial interventions will address instant blood-mediated inflammatory reaction (IBMIR), apoptosis, and inflammation, and will translate into further marked improvements in single-donor success. Effective control of auto- and alloimmunity is the key to long-term islet function, and high-resolution cellular and antibody-based assays will add considerable precision to this process. Advances in immunosuppression, with new antibody-based targeting of costimulatory blockade and other T-B cellular signaling, will have further profound impact on the safety record of immunotherapy. Clinical trials will move forward shortly to test out new human stem cell derived islets, and in parallel trials will move forward, testing pig islets for compatibility in patients. Induction of immunological tolerance to self-islet antigens and to allografts is a difficult challenge, but potentially within our grasp. PMID:23804275

Gal alpha (1,3)Gal, the major xenoantigen(s) recognised in pigs by human natural antibodies.

PubMed

Sandrin, M S; McKenzie, I F

1994-10-01

The transplantation of pig organs to humans (xenotransplantation) is now receiving serious consideration because of the shortage of human donors for organ transplants of kidney, liver and heart, and of islet cell transplantation for diabetes. The problem with such xenografts would be hyperacute rejection--mediated by natural antibodies in humans to pig antigens, complement fixation to endothelial cells, and the rapid onset of intravascular coagulation. It is now clear that the major target of the natural IgM and IgG antibodies is the terminal carbohydrate epitope Gal alpha(1,3)Gal, formed by the alpha 1,3galactosyl transferase, which places a terminal galactose residue in an alpha-linkage to another galactose. The alpha 1,3galactosyl transferase in the pig gives rise to very high endothelial cell expression of Gal alpha(1,3)Gal, a ready explanation for the hyperacute rejection of vascularized organs. In addition the parenchuma of liver and kidneys have high levels of Gal alpha-(1,3)Gal. These tissues will all fail in a pig-to-human transplant in what can now be precisely defined in terms of antigen and antibody. We have already made some suggestions for removal of anti-Gal alpha(1,3)Gal antibodies and if the procedure were technically feasible xenotransplantation could be attempted now, especially in patients doomed to a certain death because of the absence of a donor (especially for liver where ex vivo perfusion could be performed). However, the immune system is far from simple, as is shown by the healthy status of mice lacking MHC Class I, Class II or both Class I & II molecules. Perhaps the curtain is about to go up to reveal a new scene! Islets differ from the other tissues and may well not undergo acute antibody-mediated hyperacute rejection--it will be of interest to see how these fare in xenotransplantation models or even in patients. Again, normal individuals do not have anti-islet antibodies; but a proportion of diabetic patients do have such antibodies--whether these will cause hyperacute or acute rejection or are markers of immunity of T-cell type, remains to be seen. Whatever, the area is exciting, is progressing rapidly and, as indicated elsewhere, within a few years we should know whether modified pig tissue can be grafted to some patients. The isolation of the cDNA clone encoding the pig alpha 1,3 galactosyl transferase is an essential first step in the production of a transgenic pig lacking the alpha 1,3Galactosyltransferase and therefore the Gal alpha(1,3)Gal epitope, and such animals could serve as donor for human transplantation.

Hyaluronic Acid/Collagen Hydrogel as an Alternative to Alginate for Long-Term Immunoprotected Islet Transplantation.

PubMed

Harrington, Stephen; Williams, Janette; Rawal, Sonia; Ramachandran, Karthik; Stehno-Bittel, Lisa

2017-10-01

Alginate has long been the material of choice for immunoprotection of islets due to its low cost and ability to easily form microspheres. Unfortunately, this seaweed-derived material is notoriously prone to fibrotic overgrowth in vivo, resulting in premature graft failure. The purpose of this study was to test an alternative, hyaluronic acid (HA-COL), for in vitro function, viability, and allogeneic islet transplant outcomes in diabetic rats. In vitro studies indicated that the HA-COL gel had diffusion characteristics that would allow small molecules such as glucose and insulin to enter and exit the gel, whereas larger molecules (70 and 500 kDa dextrans) were impeded from diffusing past the gel edge in 24 h. Islets encapsulated in HA-COL hydrogel showed significantly improved in vitro viability over unencapsulated islets and retained their morphology and glucose sensitivity for 28 days. When unencapsulated allogeneic islet transplants were administered to the omentum of outbred rats, they initially were normoglycemic, but by 11 days returned to hyperglycemia. Immunohistological examination of the grafts and surrounding tissue indicated strong graft rejection. By comparison, when using the same outbred strain of rats, allogeneic transplantation of islets within the HA-COL gel reversed long-term diabetes and prevented graft rejection in all animals. Animals were sacrificed at 40, 52, 64, and 80 weeks for evaluation, and all were non-diabetic at sacrifice. Explanted grafts revealed viable islets in the transplant site as well as intact hydrogel, with little or no evidence of fibrotic overgrowth or cellular rejection. The results of these studies demonstrate great potential for HA-COL hydrogel as an alternative to sodium alginate for long-term immunoprotected islet transplantation.

Polyamide nanocapsules and nano-emulsions containing Parsol® MCX and Parsol® 1789: in vitro release, ex vivo skin penetration and photo-stability studies.

PubMed

Hanno, Ibrahim; Anselmi, Cecilia; Bouchemal, Kawthar

2012-02-01

To prepare polyamide nanocapsules for skin photo-protection, encapsulating α-tocopherol, Parsol®MCX (ethylhexyl methoxycinnamate) and/or Parsol®1789 (butyl methoxydibenzoylmethane). Nanocapsules were obtained by combining spontaneous emulsification and interfacial polycondensation reaction between sebacoyl chloride and diethylenetriamine. Nano-emulsions used as control were obtained by the same process without monomers. The influence of carrier on release rate was studied in vitro with a membrane-free model. Epidermal penetration of encapsulated sunscreens was ex vivo evaluated using Franz diffusion cells. Ability of encapsulated sunscreens to improve photo-stability was verified by comparing percentage of degradation after UV radiation exposure. Sunscreen-containing nanocapsules (260-400 nm) were successfully prepared; yield of encapsulation was >98%. Parsol®MCX and Parsol®1789 encapsulation led to decreased release rate by up to 60% in comparison with nano-emulsion and allowed minimum penetration through pig ear epidermis. Presence of polyamide shell protected encapsulated sunscreen filters from photo-degradation without affecting their activity. Encapsulation of Parsol®MCX and Parsol®1789 into oil-core of polyamide nanocapsules allowed protection from photo-degradation, controlled release from nanocapsules, and limited penetration through pig ear epidermis.

Effect of oxygen supply on the size of implantable islet-containing encapsulation devices.

PubMed

Papas, Klearchos K; Avgoustiniatos, Efstathios S; Suszynski, Thomas M

2016-03-01

Beta-cell replacement therapy is a promising approach for the treatment of diabetes but is currently limited by the human islet availability and by the need for systemic immunosuppression. Tissue engineering approaches that will enable the utilization of islets or β-cells from alternative sources (such as porcine islets or human stem cell derived beta cells) and minimize or eliminate the need for immunosuppression have the potential to address these critical limitations. However, tissue engineering approaches are critically hindered by the device size (similar to the size of a large flat screen television) required for efficacy in humans. The primary factor dictating the device size is the oxygen availability to islets to support their viability and function (glucose-stimulated insulin secretion [GSIS]). GSIS is affected (inhibited) at a much higher oxygen partial pressure [pO2] than that of viability (e.g. 10 mmHg as opposed to 0.1 mmHg). Enhanced oxygen supply (higher pO2) than what is available in vivo at transplant sites can have a profound effect on the required device size (potentially reduce it to the size of a postage stamp). This paper summarizes key information on the effect of oxygen on islet viability and function within immunoisolation devices and describes the potential impact of enhanced oxygen supply to devices in vivo on device size reduction.

Entrapment of dispersed pancreatic islet cells in CultiSpher-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulation.

PubMed

Del Guerra, S; Bracci, C; Nilsson, K; Belcourt, A; Kessler, L; Lupi, R; Marselli, L; De Vos, P; Marchetti, P

2001-12-20

Immunoprotection of pancreatic islets for successful allo- or xenotransplantation without chronic immunosuppression is an attractive, but still elusive, approach for curing type 1 diabetes. It was recently shown that, even in the absence of fibrotic overgrowth, other factors, mainly insufficient nutrition to the core of the islets, represent a major barrier for long-term survival of intraperitoneal microencapsulated islet grafts. The use of dispersed cells might contribute to solve this problem due to the conceivably easier nutritional support to the cells. In the present study, purified bovine islets, prepared by collagenase digestion and density gradient purification, and dispersed bovine islet cells, obtained by trypsin and DNAsi (viability > 90%), were entrapped into either 2% (w/v) sodium alginate (commonly used for encapsulation purposes) or (dispersed islet cells only) macroporous gelatin microcarriers (CulthiSpher-S, commonly used for the production of biologicals by animal cells). Insulin release studies in response to glucose were performed within 1 week and after 1 month from preparation of the varying systems and showed no capability of dispersed bovine islet cells within sodium alginate microcapsules to sense glucose concentration changes. On the contrary, bovine islet cells entrapped in CulthiSpher-S microcarriers showed maintained capacity of increasing insulin secretion upon enhanced glucose concentration challenge. In this case, insulin release was approximately 60% of that from intact bovine islets within sodium alginate microcapsules. MTT and hematoxylineosin staining of islet cell-containing microcarriers showed the presence of viable and metabolically active cells throughout the study period. This encouraging functional data prompted us to test whether the microcarriers could be immunoisolated for potential use in transplantation. The microcarriers were embedded within 3% sodium alginate, which was then covered with a poly-L-lysine layer and a final outer alginate layer. Maintained insulin secretion function of this system was observed, which raises the possibility of using microencapsulated CulthiSpher-S microcarriers, containing dispersed pancreatic islet cells, in experimental transplantation studies. Copyright 2001 John Wiley & Sons, Inc.

Engraftment and reversal of diabetes after intramuscular transplantation of neonatal porcine islet-like clusters.

PubMed

Wolf-van Buerck, Lelia; Schuster, Marion; Baehr, Andrea; Mayr, Tanja; Guethoff, Sonja; Abicht, Jan; Reichart, Bruno; Nam-Apostolopoulos, Yun-Chung; Klymiuk, Nikolai; Wolf, Eckhard; Seissler, Jochen

2015-01-01

Intraportal infusion is currently the method of choice for clinical islet cell transplantation but suffers from poor efficacy. As the liver may not represent an optimal transplantation site for Langerhans islets, we examined the potential of neonatal porcine islet-like clusters (NPICCs) to engraft in skeletal muscle as an alternative transplantation site. Neonatal porcine islet-like clusters were isolated from 2- to 5-day-old piglets and either transplanted under the kidney capsule (s.k.) or injected into the lower hindlimb muscle (i.m.) of streptozotocin-diabetic NOD-SCID IL2rγ(-/-) (NSG) mice. Survival, vascularization, maturation, and functional activity were analyzed by intraperitoneal glucose tolerance testing and immunohistochemical analyses. Intramuscular transplantation of NPICCs resulted in development of normoglycemia and restored glucose homeostasis. Time to reversal of diabetes and glucose tolerance (AUC glucose and AUC insulin) did not significantly differ as compared to s.k. transplantation. Intramuscular grafts exhibited rapid neovascularization and graft composition with cytokeratin-positive ductal cells and beta cells at post-transplant weeks 2 and 8 and after establishment of normoglycemia was comparable in both groups. Intramuscular injection represents a minimally invasive but efficient alternative for transplantation of NPICCs and, thus, offers an attractive alternative site for xenotransplantation approaches. These findings may have important implications for improving the outcome and the monitoring of pig islet xenotransplantation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Potential secondary poisoning risks to non-targets from a sodium nitrite toxic bait for invasive wild pigs.

PubMed

Snow, Nathan P; Foster, Justin A; VanNatta, Eric H; Horak, Katherine E; Humphrys, Simon T; Staples, Linton D; Hewitt, David G; VerCauteren, Kurt C

2018-01-01

An acute and orally delivered toxic bait containing micro-encapsulated sodium nitrite (MESN), is under development to provide a novel and humane technology to help curtail damage caused by invasive wild pigs (Sus scrofa). We evaluated potential secondary risks for non-target species by: testing whether four different types of micro-encapsulation coatings could reduce vomiting by invasive wild pigs, testing the levels of residual sodium nitrite (SN) in tissues of invasive wild pigs, testing the environmental persistence of SN in vomitus, and conducting a risk assessment for scavengers. Micro-encapsulation coatings did not affect the frequency of vomiting. We identified no risk of secondary poisoning for non-target scavengers that consume muscle, eyes, and livers of invasive wild pig carcasses because residual SN from the toxic bait was not detected in those tissues. The risk of secondary poisoning from consuming vomitus appeared low because ∼90% of the SN was metabolized or broken down prior to vomiting, and continued to degrade after being exposed to the environment. Secondary poisoning could occur for common scavengers that consume approximately ≥15% of their daily dietary requirements of digestive tract tissues or undigested bait from carcasses of invasive wild pigs in a rapid, single-feeding event. The likelihood of this occurring in a natural setting is unknown. The digestive tracts of poisoned invasive wild pigs contained an average of ∼4.35 mg/g of residual SN. Data from this study suggest no risks of secondary poisoning for non-target species (including humans) that consume muscle, liver, or eyes of invasive wild pigs poisoned with a MESN toxic bait. More species-specific testing for scavengers that consume digestive tract tissues and undigested bait is needed to reduce uncertainty about these potential risks. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Real-time bioluminescence imaging of macroencapsulated fibroblasts reveals allograft protection in rhesus monkeys (Macaca mulatta).

PubMed

Tarantal, Alice F; Lee, C Chang I; Itkin-Ansari, Pamela

2009-07-15

Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte device was shown to provide long-term immunoprotection of murine islets in a mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner. Encapsulated cells were transplanted subcutaneously (n=2), or cells were injected without encapsulation (n=1) and outcomes compared. BLI was performed to monitor cell survival. The BLI signal from the encapsulated cells remained robust postinsertion and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days. These data demonstrate that TheraCyte encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys.

Profile of new green fluorescent protein transgenic Jinhua pigs as an imaging source

NASA Astrophysics Data System (ADS)

Kawarasaki, Tatsuo; Uchiyama, Kazuhiko; Hirao, Atsushi; Azuma, Sadahiro; Otake, Masayoshi; Shibata, Masatoshi; Tsuchiya, Seiko; Enosawa, Shin; Takeuchi, Koichi; Konno, Kenjiro; Hakamata, Yoji; Yoshino, Hiroyuki; Wakai, Takuya; Ookawara, Shigeo; Tanaka, Hozumi; Kobayashi, Eiji; Murakami, Takashi

2009-09-01

Animal imaging sources have become an indispensable material for biological sciences. Specifically, gene-encoded biological probes serve as stable and high-performance tools to visualize cellular fate in living animals. We use a somatic cell cloning technique to create new green fluorescent protein (GFP)-expressing Jinhua pigs with a miniature body size, and characterized the expression profile in various tissues/organs and ex vivo culture conditions. The born GFP-transgenic pig demonstrate an organ/tissue-dependent expression pattern. Strong GFP expression is observed in the skeletal muscle, pancreas, heart, and kidney. Regarding cellular levels, bone-marrow-derived mesenchymal stromal cells, hepatocytes, and islet cells of the pancreas also show sufficient expression with the unique pattern. Moreover, the cloned pigs demonstrate normal growth and fertility, and the introduced GFP gene is stably transmitted to pigs in subsequent generations. The new GFP-expressing Jinhua pigs may be used as new cellular/tissue light resources for biological imaging in preclinical research fields such as tissue engineering, experimental regenerative medicine, and transplantation.

The permeability of EUDRAGIT RL and HEMA-MMA microcapsules to glucose and inulin.

PubMed

Douglas, J A; Sefton, M V

1990-10-05

Measurement of the rate of glucose diffusion from EUDGRAGIT RL and HEMA-MMA microcapsules coupled with a Thiele modulus/Biot number analysis of the glucose utilization rate suggests that pancreatic islets and CHO (Chinese hamster ovary) cells (at moderate to high cell densities) should not be adversely affected by the diffusion restrictions associated with these capsule membranes. The mass transfer coefficients for glucose at 20 degrees C were of the same order of magnitude for both capsules, based on release measurements: approximately 5 x 10(-6) cm/s for EUDRAGIT RL and approximately 2 x 10(-6) for HEMA-MMA. Inulin release from EUDRAGIT RL was slower than for glucose (mass transfer coefficient 14 +/- 4 x 10(-8) cm/s). The Thiele moduli were much less than 1, either for a single islet at the center of a capsule or CHO cells uniformly distributed throughout a capsule at 10(-6) cells/ mL, so that diffusion restrictions within the cells in EUDRAGIT RL or 800 microm HEMA-MMA capsules should be negligible. The ratio of external to internal diffusion resistance (Biot number) was less than 1, so that at most, only a small diffusion effect on glucose utilization should be expected (i.e., the overall effectiveness factors were greater than 0.8). These calculations were consistent with experimental observation of encapsulated islet behavior but not fully with CHO cell behavior. Permeability restricted cell viability and growth is potentially a major limitation of encapsulated cells; further analysis is warranted.

Encapsulation system for the immunoisolation of living cells

NASA Technical Reports Server (NTRS)

Lacik, Igor (Inventor); Brissova, Marcela (Inventor); Wang, Taylor G. (Inventor); Anikumar, Amrutur V. (Inventor); Prokop, Ales (Inventor); Powers, Alvin C. (Inventor)

1999-01-01

The present invention is drawn to a composition of matter comprising high viscosity sodium alginate, cellulose sulfate and a multi-component polycation. Additionally, the present invention provides methods for making capsules, measuring capsule permeability to immunologically-relevant proteins and treating disease in an animal using encapsulated cells. Over one thousand combinations of polyanions and polycations were examined as polymer candidates suitable for encapsulation of living cells and thirty-three pairs were effective. The combination of sodium alginate, cellulose sulfate, poly(methylene-co-guanidine) hydrochloride, calcium chloride, and sodium chloride produced the most desirable results. Pancreatic islets encapsulated in this multicomponent capsule demonstrated glucose-stimulated insulin secretion in vitro and reversed diabetes without stimulating immune reaction in mice. The capsule formulation and system of the present invention allows independent adjustments of capsule size, wall thickness, mechanical strength and permeability, and offers distinct advantages for immunoisolating cells.

Persufflation Improves Pancreas Preservation When Compared With the Two-Layer Method

PubMed Central

Scott, W.E.; O'Brien, T.D.; Ferrer-Fabrega, J.; Avgoustiniatos, E.S.; Weegman, B.P.; Anazawa, T.; Matsumoto, S.; Kirchner, V.A.; Rizzari, M.D.; Murtaugh, M.P.; Suszynski, T.M.; Aasheim, T.; Kidder, L.S.; Hammer, B.E.; Stone, S.G.; Tempelman, L.; Sutherland, D.E.R.; Hering, B.J.; Papas, K.K.

2010-01-01

Islet transplantation is emerging as a promising treatment for patients with type 1 diabetes. It is important to maximize viable islet yield for each organ due to scarcity of suitable human donor pancreata, high cost, and the high dose of islets required for insulin independence. However, organ transport for 8 hours using the two-layer method (TLM) frequently results in lower islet yields. Since efficient oxygenation of the core of larger organs (eg, pig, human) in TLM has recently come under question, we investigated oxygen persufflation as an alternative way to supply the pancreas with oxygen during preservation. Porcine pancreata were procured from non–heart-beating donors and preserved by either TLM or persufflation for 24 hours and fixed. Biopsies were collected from several regions of the pancreas, sectioned, stained with hematoxylin and eosin, and evaluated by a histologist. Persufflated tissues exhibited distended capillaries due to gas perfusion and significantly less autolysis/cell death than regions not exposed to persufflation or tissues exposed to TLM. The histology presented here suggests that after 24 hours of preservation, persufflation dramatically improves tissue health when compared with TLM. These results indicate the potential for persufflation to improve viable islet yields and extend the duration of preservation, allowing more donor organs to be utilized. PMID:20692396

Microcapsules with Intrinsic Barium Radiopacity for Immunoprotection and X-ray/CT imaging of Pancreatic Islet Cells

PubMed Central

Arifin, D.R.; Manek, S.; Call, E.; Arepally, A.; Bulte, J.W.M.

2012-01-01

Microencapsulation is a commonly used technique for immunoprotection of engrafted therapeutic cells. We investigated a library of capsule formulations to determine the most optimal formulation for pancreatic beta islet cell transplantation, using barium as the gelating ion and clinical-grade protamine sulfate (PS) as a new cationic capsule cross-linker. Barium-gelated alginate/PS/alginate microcapsules (APSA, diameter = 444±21 μm) proved to be mechanically stronger and supported a higher cell viability as compared to conventional alginate/poly-L-lysine/alginate (APLLA) capsules. Human pancreatic islets encapsulated inside APSA capsules, gelated with 20 mM barium as optimal concentration, exhibited a sustained morphological integrity, viability, and functionality for at least 3–4 weeks in vitro, with secreted human C-peptide levels of 0.2–160 pg/ml/islet. Unlike APLLA capsules that are gelled with calcium, barium-APSA capsules are intrinsically radiopaque and, when engrafted into mice, could be readily imaged in vivo with micro-computed tomography (CT). Without the need of adding contrast agents, these capsules offer a clinically applicable alternative for simultaneous immunoprotection and real-time, non-invasive X-ray/CT monitoring of engrafted cells during and after in vivo administration. PMID:22444642

Long-term follow-up of patients with type 1 diabetes transplanted with neonatal pig islets

PubMed Central

Valdes-Gonzalez, R; Rodriguez-Ventura, A L; White, D J G; Bracho-Blanchet, E; Castillo, A; Ramírez-González, B; López-Santos, M G; León-Mancilla, B H; Dorantes, L M

2010-01-01

Pancreas transplantation is an option to achieve better metabolic control and decrease chronic complications in patients with diabetes. Xenotransplantation becomes an important alternative. In this study, we show the clinical outcome of patients with type 1 diabetes transplanted with neonatal pig islets without immunosuppression. In a longitudinal study of 23 patients with type 1 diabetes, who received porcine islets between 2000 and 2004, we registered demographic and clinical characteristics every 3 months and chronic complications evaluation yearly. Porcine C-peptide was measured in urine samples under basal conditions and after stimulation with l-arginine. More than 50% were female, median current age was 20·8 years, median diabetes duration at transplantation 5·5 years, median current diabetes duration 11 years and median time post-transplantation 5·7 years. Their media of glycosylated haemoglobin reduced significantly after the first transplantation. Insulin doses remain with a reduction greater than 33% in more than 50% of the patients. Before transplantation, 14 of the 21 patients presented mild chronic complications and currently only two patients presented these complications. Porcine C-peptide was present in all urine samples under basal conditions and increased post-stimulation with l-arginine. These patients achieved an excellent metabolic control after the first transplantation. This could explain, as well as the remaining function of transplanted cells, the low frequency of chronic complications compared to patients with similar diabetes duration and age. PMID:20964645

Long-term follow-up of patients with type 1 diabetes transplanted with neonatal pig islets.

PubMed

Valdes-Gonzalez, R; Rodriguez-Ventura, A L; White, D J G; Bracho-Blanchet, E; Castillo, A; Ramírez-González, B; López-Santos, M G; León-Mancilla, B H; Dorantes, L M

2010-12-01

Pancreas transplantation is an option to achieve better metabolic control and decrease chronic complications in patients with diabetes. Xenotransplantation becomes an important alternative. In this study, we show the clinical outcome of patients with type 1 diabetes transplanted with neonatal pig islets without immunosuppression. In a longitudinal study of 23 patients with type 1 diabetes, who received porcine islets between 2000 and 2004, we registered demographic and clinical characteristics every 3 months and chronic complications evaluation yearly. Porcine C-peptide was measured in urine samples under basal conditions and after stimulation with l-arginine. More than 50% were female, median current age was 20·8 years, median diabetes duration at transplantation 5·5 years, median current diabetes duration 11 years and median time post-transplantation 5·7 years. Their media of glycosylated haemoglobin reduced significantly after the first transplantation. Insulin doses remain with a reduction greater than 33% in more than 50% of the patients. Before transplantation, 14 of the 21 patients presented mild chronic complications and currently only two patients presented these complications. Porcine C-peptide was present in all urine samples under basal conditions and increased post-stimulation with l-arginine. These patients achieved an excellent metabolic control after the first transplantation. This could explain, as well as the remaining function of transplanted cells, the low frequency of chronic complications compared to patients with similar diabetes duration and age. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

Diffusion coefficient of alginate microcapsules used in pancreatic islet transplantation, a method to cure type 1 diabetes

NASA Astrophysics Data System (ADS)

Najdahmadi, Avid; Lakey, Jonathan R. T.; Botvinick, Elliot

2018-02-01

Pancreatic islet transplantation is a promising approach of providing insulin in type 1 diabetes. One strategy to protect islets from the host immune system is encapsulation within a porous biocompatible alginate membrane. This encapsulation provides mechanical support to the cells and allows selective diffusion of oxygen, nutrients and insulin while blocking immunoglobulins. These hydrogels form by diffusion of calcium ions into the polymer network and therefore they are highly sensitive to environmental changes and fluctuations in temperature. We investigated the effects of gel concentration, crosslinking time and ambient conditions on material permeability, volume, and rigidity, all of which may change the immunoisolating characteristics of alginate. To measure diffusion coefficient as a method to capture structural changes we studied the diffusion of fluorescently tagged dextrans of different molecular weight into the midplane of alginate microcapsules, the diffusion coefficient is then calculated by fitting observed fluorescence dynamics to the mathematical solution of 1-D diffusion into a sphere. These measurements were performed after incubation in different conditions as well as after an in vivo experiment in six immunocompetent mice for seven days. Additionally, the changes in gel volume after incubation at different temperatures and environmental conditions as well as changes in compression modulus of alginate gels during crosslinking were investigated. Our result show that increase of polymer concentration and crosslinking time leads to a decrease in volume and increase in compression modulus. Furthermore, we found that samples crosslinked and placed in physiological environment, experience an increase in volume. As expected, these volume changes affect diffusion rates of fluorescent dextrans, where volume expansion is correlated with higher calculated diffusion coefficient. This observation is critical to islet protection since higher permeability due to the expansion in vivo may lead to increased permeability to immunoglobulins. Capsules from the in vivo study showed similar volume expansion and increased permeability, indicating our in vitro assay is a good predictor of volume change in vivo.

Assessment of pancreas cells

NASA Technical Reports Server (NTRS)

Vanoss, C. J.

1978-01-01

Pancreatic islets were obtained from guinea pig pancreas by the collagenase method and kept alive in tissue culture prior to further studies. Pancreas cell morphology was studied by standard histochemical techniques using light microscopy. Preparative vertical electrophoresis-levitation of dispersed fetal guinea pig pancreas cells was conducted in phosphate buffer containing a heavy water (D20) gradient which does not cause clumping of cells or alter the osmolarity of the buffers. The faster migrating fractions tended to be enriched in beta-cell content. Alpha and delta cells were found to some degree in most fractions. A histogram showing the cell count distribution is included.

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes--Chapter 3: Porcine islet product manufacturing and release testing criteria.

PubMed

Rayat, Gina R; Gazda, Lawrence S; Hawthorne, Wayne J; Hering, Bernhard J; Hosking, Peter; Matsumoto, Shinichi; Rajotte, Ray V

2016-01-01

In the 2009 IXA consensus, the requirements for the quality and control of manufacturing of porcine islet products were based on the U.S. regulatory framework where the porcine islet products fall within the definition of somatic cell therapy under the statutory authority of the U.S. Food and Drug Administration (FDA). In addition, porcine islet products require pre-market approval as a biologic product under the Public Health Services Act and they meet the definition of a drug under the Federal Food, Drug, and Cosmetic Act (FD&C Act). Thus, they are subject to applicable provisions of the law and as such, control of manufacturing as well as reproducibility and consistency of porcine islet products, safety of porcine islet products, and characterization of porcine islet products must be met before proceeding to clinical trials. In terms of control of manufacturing as well as reproducibility and consistency of porcine islet products, the manufacturing facility must be in compliance with current Good Manufacturing Practices (cGMP) guidelines appropriate for the initiation of Phase 1/2 clinical trials. Sponsors intending to conduct a Phase 1/2 trial of islet xenotransplantation products must be able to demonstrate the safety of the product through the establishment of particular quality assurance and quality control procedures. All materials (including animal source and pancreas) used in the manufacturing process of the porcine islet products must be free of adventitious agents. The final porcine islet product must undergo tests for the presence of these adventitious agents including sterility, mycoplasma (if they are cultured), and endotoxin. Assessments of the final product must include the safety specifications mentioned above even if the results are not available until after release as these data would be useful for patient diagnosis and treatment if necessary. In addition, a plan of action must be in place for patient notification and treatment in case the sterility culture results are positive. In terms of the characterization of porcine islet products and product release criteria, the information on the porcine islet products should be acquired from a sample of the final product to be used for transplantation and must include the morphology of the islets, specific identity, purity, viability, and potency of the product. In addition, information on the quantity of the islet products should also be provided in a standardized fashion and this should be in terms of islet equivalents and/or cell numbers. The current consensus was created to provide guidelines that manufacturing facilities may find helpful in the manufacture of and the release criteria for porcine islet products including encapsulated islets and combined islet products. Our intent with the above recommendations is to provide a framework for individual porcine islet manufacturing facilities to ensure a high level of safety for the initiation of Phase 1/2 clinical trials on porcine islet xenotransplantation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Real-time Bioluminescence Imaging of Macroencapsulated Fibroblasts Reveals Allograft Protection in Rhesus Monkeys (Macaca mulatta)

PubMed Central

Tarantal, Alice F.; Lee, C. Chang I.; Itkin-Ansari, Pamela

2009-01-01

Background Encapsulation of cells has the potential to eliminate the need for immunosuppression for cellular transplantation. Recently, the TheraCyte® device was shown to provide long-term immunoprotection of murine islets in the NOD/SCID mouse model of diabetes. In this report, translational studies were undertaken using skin fibroblasts from an unrelated rhesus monkey donor that were transduced with an HIV-1-derived lentiviral vector expressing firefly luciferase permitting the use of bioluminescence imaging (BLI) to monitor cell survival over time and in a noninvasive manner. Methods Encapsulated cells were transplanted subcutaneously (N=2) or cells were injected without encapsulation (N=1) and outcomes compared. BLI was performed to monitor cell survival. Results The BLI signal from the encapsulated cells remained robust post-insertion, and in one animal persisted for up to 1 year. In contrast, the control animal that received unencapsulated cells exhibited a complete loss of cell signal within 14 days. Conclusions These data demonstrate that TheraCyte® encapsulation of allogeneic cells provides robust immune protection in transplanted rhesus monkeys. PMID:19584678

Microcapsules with intrinsic barium radiopacity for immunoprotection and X-ray/CT imaging of pancreatic islet cells.

PubMed

Arifin, Dian R; Manek, Sameer; Call, Emma; Arepally, Aravind; Bulte, Jeff W M

2012-06-01

Microencapsulation is a commonly used technique for immunoprotection of engrafted therapeutic cells. We investigated a library of capsule formulations to determine the most optimal formulation for pancreatic beta islet cell transplantation, using barium as the gelating ion and clinical-grade protamine sulfate (PS) as a new cationic capsule cross-linker. Barium-gelated alginate/PS/alginate microcapsules (APSA, diameter = 444 ± 21 μm) proved to be mechanically stronger and supported a higher cell viability as compared to conventional alginate/poly-l-lysine/alginate (APLLA) capsules. Human pancreatic islets encapsulated inside APSA capsules, gelated with 20 mm barium as optimal concentration, exhibited a sustained morphological integrity, viability, and functionality for at least 3-4 weeks in vitro, with secreted human C-peptide levels of 0.2-160 pg/ml/islet. Unlike APLLA capsules that are gelled with calcium, barium-APSA capsules are intrinsically radiopaque and, when engrafted into mice, could be readily imaged in vivo with micro-computed tomography (CT). Without the need of adding contrast agents, these capsules offer a clinically applicable alternative for simultaneous immunoprotection and real-time, non-invasive X-ray/CT monitoring of engrafted cells during and after in vivo administration. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed

Carlsson, Per-Ola; Espes, Daniel; Sedigh, Amir; Rotem, Avi; Zimerman, Baruch; Grinberg, Helena; Goldman, Tali; Barkai, Uriel; Avni, Yuval; Westermark, Gunilla T; Carlbom, Lina; Ahlström, Håkan; Eriksson, Olof; Olerud, Johan; Korsgren, Olle

2017-12-29

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). © 2018 The Authors. American Journal of Transplantation published by Wiley Periodicals, Inc. on behalf of The American Society of Transplantation and the American Society of Transplant Surgeons.

Evaluation of Encapsulated Liver Cell Spheroids in a Fluidised-Bed Bioartificial Liver for Treatment of Ischaemic Acute Liver Failure in Pigs in a Translational Setting

PubMed Central

Selden, Clare; Spearman, Catherine Wendy; Kahn, Delawir; Miller, Malcolm; Figaji, Anthony; Erro, Eloy; Bundy, James; Massie, Isobel; Chalmers, Sherri-Ann; Arendse, Hiram; Gautier, Aude; Sharratt, Peter; Fuller, Barry; Hodgson, Humphrey

2013-01-01

Liver failure is an increasing problem. Donor-organ shortage results in patients dying before receiving a transplant. Since the liver can regenerate, alternative therapies providing temporary liver-support are sought. A bioartificial-liver would temporarily substitute function in liver failure buying time for liver regeneration/organ-procurement. Our aim: to develop a prototype bioartificial-liver-machine (BAL) comprising a human liver-derived cell-line, cultured to phenotypic competence and deliverable in a clinical setting to sites distant from its preparation. The objective of this study was to determine whether its use would improve functional parameters of liver failure in pigs with acute liver failure, to provide proof-of-principle. HepG2cells encapsulated in alginate-beads, proliferated in a fluidised-bed-bioreactor providing a biomass of 4–6×1010cells, were transported from preparation-laboratory to point-of-use operating theatre (6000miles) under perfluorodecalin at ambient temperature. Irreversible ischaemic liver failure was induced in anaesthetised pigs, after portal-systemic-shunt, by hepatic-artery-ligation. Biochemical parameters, intracranial pressure, and functional-clotting were measured in animals connected in an extracorporeal bioartificial-liver circuit. Efficacy was demonstrated comparing outcomes between animals connected to a circuit containing alginate-encapsulated cells (Cell-bead BAL), and those connected to circuit containing alginate capsules without cells (Empty-bead BAL). Cells of the biomass met regulatory standards for sterility and provenance. All animals developed progressive liver-failure after ischaemia induction. Efficacy of BAL was demonstrated since animals connected to a functional biomass (+ cells) had significantly smaller rises in intracranial pressure, lower ammonia levels, more bilirubin conjugation, improved acidosis and clotting restoration compared to animals connected to the circuit without cells. In the +cell group, human proteins accumulated in pigs' plasma. Delivery of biomass using a short-term cold-chain enabled transport and use without loss of function over 3days. Thus, a fluidised-bed bioreactor containing alginate-encapsulated HepG2cell-spheroids improved important parameters of acute liver failure in pigs. The system can readily be up-scaled and transported to point-of-use justifying development at clinical scale. PMID:24367515

Bibliometric analysis of the top-cited articles on islet transplantation

PubMed Central

Pu, Qiang-Hong; Lyu, Qiu-Ju; Liu, Huan; Fan, Kai-Hua

2017-01-01

Abstract Aims: To identify and characterize the top-cited articles in the field of islet transplantation. Methods: We used the Science Citation Index Expanded database to identify the most frequently cited articles published after 1900. Articles were evaluated using the following characteristics: citation number, publication year, study design, references, country and institution of origin, authorship, and journal. Keyword analysis and citation networks were used to analyze research trends. Results: The most frequently cited articles received between 146 and 2988 citations; the median was 291. All of the most frequently cited articles were published between 1972 and 2012, and 85 articles were published after 1990. The most popular study design involved basic science (75 articles). The leading countries were the United States (US) and Canada, and the leading institutions were the University of Alberta, Canada, and the University of Minnesota, in the US. Journals specializing in diabetes or transplantation published more than half of the articles (n = 53, 52%), with the journal Diabetes publishing the largest number (n = 30). No association was found between a journal's impact factor and the number of top-cited articles it published. There was no correlation between the number of citations and the number of years since publication, authors, participating institutions, or countries involved. Top-cited articles focused on 2 themes: the use of antirejection immunotherapy or biocompatible encapsulations to prolong graft survival, and assessments of the efficacy of islet transplants, in particular, islet allografts. Conclusions: Our study can help researchers to identify and decipher the characteristics of top-cited articles in the field of islet transplantation. Just as clinically successful allografts are carried out using the Edmonton protocol, autografts and xenografts should be similarly strengthened to solve problems relating to immune rejection and islet sources, respectively. PMID:29095254

Bibliometric analysis of the top-cited articles on islet transplantation.

PubMed

Pu, Qiang-Hong; Lyu, Qiu-Ju; Liu, Huan; Fan, Kai-Hua

2017-11-01

To identify and characterize the top-cited articles in the field of islet transplantation. We used the Science Citation Index Expanded database to identify the most frequently cited articles published after 1900. Articles were evaluated using the following characteristics: citation number, publication year, study design, references, country and institution of origin, authorship, and journal. Keyword analysis and citation networks were used to analyze research trends. The most frequently cited articles received between 146 and 2988 citations; the median was 291. All of the most frequently cited articles were published between 1972 and 2012, and 85 articles were published after 1990. The most popular study design involved basic science (75 articles). The leading countries were the United States (US) and Canada, and the leading institutions were the University of Alberta, Canada, and the University of Minnesota, in the US. Journals specializing in diabetes or transplantation published more than half of the articles (n = 53, 52%), with the journal Diabetes publishing the largest number (n = 30). No association was found between a journal's impact factor and the number of top-cited articles it published. There was no correlation between the number of citations and the number of years since publication, authors, participating institutions, or countries involved. Top-cited articles focused on 2 themes: the use of antirejection immunotherapy or biocompatible encapsulations to prolong graft survival, and assessments of the efficacy of islet transplants, in particular, islet allografts. Our study can help researchers to identify and decipher the characteristics of top-cited articles in the field of islet transplantation. Just as clinically successful allografts are carried out using the Edmonton protocol, autografts and xenografts should be similarly strengthened to solve problems relating to immune rejection and islet sources, respectively.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats.

PubMed

Matveyenko, Aleksey V; Georgia, Senta; Bhushan, Anil; Butler, Peter C

2010-11-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant.

Inconsistent formation and nonfunction of insulin-positive cells from pancreatic endoderm derived from human embryonic stem cells in athymic nude rats

PubMed Central

Matveyenko, Aleksey V.; Georgia, Senta; Bhushan, Anil

2010-01-01

Embryonic stem cell therapy has been proposed as a therapeutic strategy to restore β-cell mass and function in T1DM. Recently, a group from Novocell (now ViaCyte) reported successful development of glucose-responsive islet-like structures after implantation of pancreatic endoderm (PE) derived from human embryonic stem cells (hESC) into immune-deficient mice. Our objective was to determine whether implantation of hESC-derived pancreatic endoderm from Novocell into athymic nude rats results in development of viable glucose-responsive pancreatic endocrine tissue. Athymic nude rats were implanted with PE derived from hESC either via implantation into the epididymal fat pads or by subcutaneous implantation into TheraCyte encapsulation devices for 20 wk. Blood glucose, weight, and human insulin/C-peptide secretion were monitored by weekly blood draws. Graft β-cell function was assessed by a glucose tolerance test, and graft morphology was assessed by immunohistochemistry and immunofluorescence. At 20 wk postimplantation, epididymal fat-implanted PE progressed to develop islet-like structures in 50% of implants, with a mean β-cell fractional area of 0.8 ± 0.3%. Human C-peptide and insulin were detectable, but at very low levels (C-peptide = 50 ± 26 pmol/l and insulin = 15 ± 7 pmol/l); however, there was no increase in human C-peptide/insulin levels after glucose challenge. There was no development of viable pancreatic tissue or meaningful secretory function when human PE was implanted in the TheraCyte encapsulation devices. These data confirm that islet-like structures develop from hESC differentiated to PE by the protocol developed by NovoCell. However, the extent of endocrine cell formation and secretory function is not yet sufficient to be clinically relevant. PMID:20587750

Prolonged survival and improved glycemia in BioBreeding diabetic rats after early sustained exposure to glucagon-like peptide 1.

PubMed

Yanay, Ofer; Moralejo, Daniel; Kernan, Kelly; Brzezinski, Margaret; Fuller, Jessica M; Barton, Randall W; Lernmark, Ake; Osborne, William R

2010-06-01

Type 1 diabetes (T1D) in both humans and BioBreeding (BB) rats is an autoimmune disease that results in complete destruction of islets and insulin dependency for life. Glucagon-like peptide 1 (GLP-1) promotes beta cell proliferation and neogenesis and has a potent insulinotropic effect. We hypothesized that the expression of GLP-1 before disease onset would increase islet mass, delay diabetes and prolong survival of BB rats. Vascular smooth muscle cells retrovirally transduced to secrete GLP-1 were seeded into TheraCyte encapsulation devices, implanted subcutaneously, and rats were monitored for diabetes. In untreated control rats, plasma GLP-1 levels were 34.5-39.5 pmol/l, whereas, in treated rats, plasma levels were elevated, in the range 90-250.4 pmol/l. Hypoglycemia was not detected and this was anticipated from the glucose-regulated action of GLP-1. Diabetes onset (mean + or - SEM) in untreated rats occurred at 56.5 + or - 0.6 days (n = 6) and, in GLP-1-treated rats, was delayed until 76.4 + or - 3.3 days (n = 5) (p < 0.001). After disease onset, untreated control rats showed a rapid weight loss and elevated blood glucose (>650 mg/dl) and did not survive beyond 11 days. At 5 days after diabetes onset, insulin-secreting islets were absent in untreated rats. By contrast, treated rats maintained weight for up to 143 days of age and showed insulin-secreting beta cells. Sustained GLP-1 expression delivered by encapsulated cells before diabetes onset in BB rats showed an improved clinical outcome, suggesting the potential for treating patients using long lasting GLP-1 analogs.

A closed system for islet isolation and purification using the COBE2991 cell processor may reduce the need of clean room facilities.

PubMed

Klaffschenkel, R A; Biesemeier, A; Waidmann, M; Northoff, H; Steurer, W; Königsrainer, A; Lembert, N

2007-01-01

During the isolation of human islets of Langerhans the digest has repeated direct contact with the ambient atmosphere. In order to fulfill GMP requirements in clinical applications, the entire cell preparation must be performed in clean room facilities. We hypothesized that the use of a closed system, which avoids the direct exposure of tissue to the atmosphere, would significantly ease the preparation procedure. To avoid the direct atmosphere exposure we tested a modification of the isolation and purification process by performing all islet preparation steps in a closed system. In this study we compared the isolation outcome of the traditional open preparation technique with the new closed system. Pancreata from 6-month-old hybrid pigs were procured in the local slaughterhouse. After digestion/filtration the digest was cooled, collected, and concentrated in centrifugation containers and purified thereafter in the COBE2991 by top loading (control). In the control group 502 +/- 253 IEQ per gram pancreas were purified. The total preparation time amounted to 12 h. In the closed system the digest was cooled and directly pumped into the COBE2991 for centrifugation followed by supernatant expelling. Bag filling, centrifugation, and expelling were repeated several times. Islets in pellet form were then purified by adding a gradient (bottom loading). Using this closed system 1098 +/- 489 IEQ per gram pancreas were purified with a total cell viability of 67 +/- 10% and a beta-cell viability of 41 +/- 13%. The total preparation time reduced to 6 h. After 24 h of cell culture the viability of beta-cells was still 56 +/- 10% and was only reduced after the addition of proapoptotic IL-1 and TNF-alpha to 40 +/- 4%, indicating that freshly isolated islets are not apoptotic. In conclusion, the closed system preparation is much faster, more effective, and less expensive than the traditional islet preparation. The closed system may be applicable for human islets preparations to restrict the need of clean room facilities for islet preparations to a minimum and may open the way for islet preparations without clean room demand.

Large scale isolation, growth, and function of porcine neonatal islet cells.

PubMed Central

Korbutt, G S; Elliott, J F; Ao, Z; Smith, D K; Warnock, G L; Rajotte, R V

1996-01-01

Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition. After 9 d of in vitro culture, tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%). During the culture period, the total beta cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9. Transplantation of grafts consisting of 3 x 10(5) beta cells (1,000 aggregated) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10(5) beta cells (2,000 aggregates) achieved euglycemia within 8 wk posttransplantation. Nephrectomy of the graft bearing kidney at 14 wk posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells. The cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation. These results indicate that the neonatal porcine pancrease can be used as a source of large numbers of viable islet cells, which have the potential for growth both in vitro and in vivo, and exhibit the metabolic capacity to correct diabetes in nude mice. PMID:8621802

Composition and function of macroencapsulated human embryonic stem cell-derived implants: comparison with clinical human islet cell grafts.

PubMed

Motté, Evi; Szepessy, Edit; Suenens, Krista; Stangé, Geert; Bomans, Myriam; Jacobs-Tulleneers-Thevissen, Daniel; Ling, Zhidong; Kroon, Evert; Pipeleers, Daniel

2014-11-01

β-Cells generated from large-scale sources can overcome current shortages in clinical islet cell grafts provided that they adequately respond to metabolic variations. Pancreatic (non)endocrine cells can develop from human embryonic stem (huES) cells following in vitro derivation to pancreatic endoderm (PE) that is subsequently implanted in immune-incompetent mice for further differentiation. Encapsulation of PE increases the proportion of endocrine cells in subcutaneous implants, with enrichment in β-cells when they are placed in TheraCyte-macrodevices and predominantly α-cells when they are alginate-microencapsulated. At posttransplant (PT) weeks 20-30, macroencapsulated huES implants presented higher glucose-responsive plasma C-peptide levels and a lower proinsulin-over-C-peptide ratio than human islet cell implants under the kidney capsule. Their ex vivo analysis showed the presence of single-hormone-positive α- and β-cells that exhibited rapid secretory responses to increasing and decreasing glucose concentrations, similar to isolated human islet cells. However, their insulin secretory amplitude was lower, which was attributed in part to a lower cellular hormone content; it was associated with a lower glucose-induced insulin biosynthesis, but not with lower glucagon-induced stimulation, which together is compatible with an immature functional state of the huES-derived β-cells at PT weeks 20-30. These data support the therapeutic potential of macroencapsulated huES implants but indicate the need for further functional analysis. Their comparison with clinical-grade human islet cell grafts sets references for future development and clinical translation. Copyright © 2014 the American Physiological Society.

Effect of strategic administration of an encapsulated blend of formic acid, citric acid, and essential oils on Salmonella carriage, seroprevalence, and growth of finishing pigs.

PubMed

Walia, Kavita; Argüello, Hector; Lynch, Helen; Leonard, Finola C; Grant, Jim; Yearsley, Dermot; Kelly, Sinead; Duffy, Geraldine; Gardiner, Gillian E; Lawlor, Peadar G

2017-02-01

Controlling Salmonella at farm level can act as the first line of defence in reducing salmonellosis from pork. This study investigated the efficacy of an encapsulated blend of formic acid, citric acid, and essential oils (FormaXOL™) administered to finisher pigs for 28days prior to slaughter in controlling Salmonella shedding on a commercial farm with a history of high Salmonella seroprevalence. Fourteen pens of 8-10 pigs/pen were randomly assigned to a control (finisher diet without additive) or a treatment group (the same diet with 4kg/t of FormaXOL™) for 28 days. Faeces were collected from each pig on days 0, 14, and 28, while on day 29 blood, caecal digesta and ileocaecal-mesenteric lymph nodes were collected at slaughter. Pigs were weighed at the start and end of the trial, feed intake was recorded, and carcass quality parameters were recorded at slaughter. On day 14, Salmonella shedding was reduced in the treatment compared to the control group (27.9% versus 51.7% probability of detecting Salmonella in faeces, respectively; p=0.001). However, on day 28, no reduction was observed (20.6% versus 35.9% probability of detecting Salmonella in faeces, respectively; p=0.07). Interestingly, Salmonella shedding rates in the treated pigs remained stable throughout the trial compared to the control group. This suggests that the feed additive prevented additional pigs from acquiring the Salmonella infection. A lower Salmonella seroprevalence was detected at slaughter in the treatment compared to the control group using the 40% optical density cut-off (64.5% versus 88.5%, respectively; p=0.01). However, no significant differences in Salmonella recovery rates were observed in the caecal digesta or lymph nodes between treated and control groups. Treated pigs had a lower feed intake than pigs fed the control diet (p=0.001); however, average daily gain and feed conversion efficiency were not affected by treatment (p=0.45 and 0.55, respectively). Consequently, supplementing the diet with FormaXOL™ for 28days increased the feed cost per kg of live-weight gain by €0.08. Overall, results suggest that strategic administration of an encapsulated blend of formic acid, citric acid, and essential oils, to finishing pigs for 28days prior to slaughter has potential to prevent increased Salmonella shedding at certain time points as well as seroprevalence. However, this additive did not lower intestinal carriage, nor did it reduce seroprevalence to below the cut-off used for the high Salmonella risk category in Ireland (50%) or improve growth performance. Copyright © 2016 Elsevier B.V. All rights reserved.

Polyanhydride nanovaccine against swine influenza virus in pigs.

PubMed

Dhakal, Santosh; Goodman, Jonathan; Bondra, Kathryn; Lakshmanappa, Yashavanth S; Hiremath, Jagadish; Shyu, Duan-Liang; Ouyang, Kang; Kang, Kyung-Il; Krakowka, Steven; Wannemuehler, Michael J; Won Lee, Chang; Narasimhan, Balaji; Renukaradhya, Gourapura J

2017-02-22

We have recently demonstrated the effectiveness of an influenza A virus (IAV) subunit vaccine based on biodegradable polyanhydride nanoparticles delivery in mice. In the present study, we evaluated the efficacy of ∼200nm polyanhydride nanoparticles encapsulating inactivated swine influenza A virus (SwIAV) as a vaccine to induce protective immunity against a heterologous IAV challenge in pigs. Nursery pigs were vaccinated intranasally twice with inactivated SwIAV H1N2 (KAg) or polyanhydride nanoparticle-encapsulated KAg (KAg nanovaccine), and efficacy was evaluated against a heterologous zoonotic virulent SwIAV H1N1 challenge. Pigs were monitored for fever daily. Local and systemic antibody responses, antigen-specific proliferation of peripheral blood mononuclear cells, gross and microscopic lung lesions, and virus load in the respiratory tract were compared among the groups of animals. Our pre-challenge results indicated that KAg nanovaccine induced virus-specific lymphocyte proliferation and increased the frequency of CD4 + CD8αα + T helper and CD8 + cytotoxic T cells in peripheral blood mononuclear cells. KAg nanovaccine-immunized pigs were protected from fever following SwIAV challenge. In addition, pigs immunized with the KAg nanovaccine presented with lower viral antigens in lung sections and had 6 to 8-fold reduction in nasal shedding of SwIAV four days post-challenge compared to control animals. Immunologically, increased IFN-γ secreting T lymphocyte populations against both the vaccine and challenge viruses were detected in KAg nanovaccine-immunized pigs compared to the animals immunized with KAg alone. However, in the KAg nanovaccine-immunized pigs, hemagglutination inhibition, IgG and IgA antibody responses, and virus neutralization titers were comparable to that in the animals immunized with KAg alone. Overall, our data indicated that intranasal delivery of polyanhydride-based SwIAV nanovaccine augmented antigen-specific cellular immune response in pigs, with promise to induce cross-protective immunity. Copyright © 2017 Elsevier Ltd. All rights reserved.

Getting the most from microfluidic platforms for biomedical applications (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shen, Amy

2016-03-01

Microfluidics has emerged in recent years as a versatile method of manipulating fluids at small length-scales, and in particular, for generating and manipulating micron size droplets with controllable size and functionality. For example, many research groups developed microfluidics devices for cell encapsulation, and synthesizing functionalized polymer microspheres and inorganic nanoparticles with precise control over their shapes and sizes. In this talk, I will showcase 2 microfluidic platforms to highlight their versatility and potential biomedical applications. (1) Droplet microfluidic platforms (a) A droplet microfluidics method to fabricate alginate microspheres while simultaneously immobilizing anti-Mycobacterium tuberculosis complex IgY and anti-Escherichia coli IgG antibodies primarily on the porous alginate carriers for specific binding and binding affinity tests. The binding affinity of antibodies is directly measured by fluorescence intensity of stained target bacteria on the microspheres. We demonstrate that the functionalized alginate microspheres yield specificity comparable with an enzyme-linked immunosorbent assay. We can easily modify the size and shape of alginate microspheres, and increase the concentration of functionalized alginate microspheres to further enhance binding kinetics and enable multiplexing. (b) A novel droplet microfluidics method to image oxygen in single islets (pancreatic cells) for glucose sensing. Individual islets and a fluorescent oxygen-sensitive dye were encased within a thin alginate polymer microcapsule for insulin secretion monitoring. The sensing system operated similarly from 2-48 hours following encapsulation, and viability and function of the islets were not significantly affected by the encapsulation process. This approach should be applicable to other cell types and dyes sensitive to other biologically important molecules. (2) A microfluidic chamber to perform uniform electric field stimulation in circular shaped culturewares A 3D computer-aided designed (CAD) polymeric insert is designed and retrofitted to circular shaped culturewares in an integrated microfluidic electrical stimulation platform to generate uniform EF with higher cell yields. In particular, NIH/3T3 mouse embryonic fibroblast cells are used to validate the performance of the 3D designed Poly(methyl methacrylate) (PMMA) inserts in a circular-shaped 6-well plate. The CAD based inserts can be easily scaled up to further increase effective stimulation area percentages, and also be implemented in commercially available culturewares for a wide variety of EF-related research such as EF-cell interaction and tissue regeneration studies.

Encapsulation of sex sorted boar semen: sperm membrane status and oocyte penetration parameters.

PubMed

Spinaci, Marcella; Chlapanidas, Theodora; Bucci, Diego; Vallorani, Claudia; Perteghella, Sara; Lucconi, Giulia; Communod, Ricardo; Vigo, Daniele; Galeati, Giovanna; Faustini, Massimo; Torre, Maria Luisa

2013-03-01

Although sorted semen is experimentally used for artificial, intrauterine, and intratubal insemination and in vitro fertilization, its commercial application in swine species is still far from a reality. This is because of the low sort rate and the large number of sperm required for routine artificial insemination in the pig, compared with other production animals, and the greater susceptibility of porcine spermatozoa to stress induced by the different sex sorting steps and the postsorting handling protocols. The encapsulation technology could overcome this limitation in vivo, protecting and allowing the slow release of low-dose sorted semen. The aim of this work was to evaluate the impact of the encapsulation process on viability, acrosome integrity, and on the in vitro fertilizing potential of sorted boar semen. Our results indicate that the encapsulation technique does not damage boar sorted semen; in fact, during a 72-hour storage, no differences were observed between liquid-stored sorted semen and encapsulated sorted semen in terms of plasma membrane (39.98 ± 14.38% vs. 44.32 ± 11.72%, respectively) and acrosome integrity (74.32 ± 12.17% vs. 66.07 ± 10.83%, respectively). Encapsulated sorted spermatozoa presented a lower penetration potential than nonencapsulated ones (47.02% vs. 24.57%, respectively, P < 0.0001), and a significant reduction of polyspermic fertilization (60.76% vs. 36.43%, respectively, polyspermic ova/total ova; P < 0.0001). However, no difference (P > 0.05) was observed in terms of total efficiency of fertilization expressed as normospermic oocytes/total oocytes (18.45% vs. 15.43% for sorted diluted and sorted encapsulated semen, respectively). The encapsulation could be an alternative method of storing of pig sex sorted spermatozoa and is potentially a promising technique in order to optimize the use of low dose of sexed spermatozoa in vivo. Copyright © 2013 Elsevier Inc. All rights reserved.

Manufacturing porcine islets: culture at 22°C has no advantage above culture at 37°C

PubMed Central

Mueller, Kate R; Martins, Kyra V; Murtaugh, Michael P; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

Background The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37°C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Methods Porcine islets were isolated from three donors and cultured at 37°C for 1 day, and then under three different conditions: 37°C for 6 days (condition A); 22°C for 6 days (condition B); or 22°C for 5 days followed by 37°C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Results Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129–159 nmol/min.mgDNA before culture, and 259–291, 204–212, and 207–228 nmol/min•mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular most of lymphocyte markers showed a >10-fold reduction and also 6 markers in the lymphokine and chemokine series: these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 hours at 37°C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22°C to those in islets cultured at 37°C. Results were consistent among the preparations from the three donors. Conclusions Culture of porcine islets at 37°C provides benefits over culture at 22°C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress. PMID:23941232

Species-specific vesicular monoamine transporter 2 (VMAT2) expression in mammalian pancreatic beta cells: implications for optimising radioligand-based human beta cell mass (BCM) imaging in animal models

PubMed Central

Hartwig, N. R.; Kalmbach, N.; Klietz, M.; Anlauf, M.; Eiden, L. E.; Weihe, E.

2014-01-01

Aims/hypothesis Imaging of beta cell mass (BCM) is a major challenge in diabetes research. The vesicular monoamine transporter 2 (VMAT2) is abundantly expressed in human beta cells. Radiolabelled analogues of tetrabenazine (TBZ; a low-molecular-weight, cell-permeant VMAT2-selective ligand) have been employed for pancreatic islet imaging in humans. Since reports on TBZ-based VMAT2 imaging in rodent pancreas have been fraught with confusion, we compared VMAT2 gene expression patterns in the mouse, rat, pig and human pancreas, to identify appropriate animal models with which to further validate and optimise TBZ imaging in humans. Methods We used a panel of highly sensitive VMAT2 antibodies developed against equivalently antigenic regions of the transporter from each species in combination with immunostaining for insulin and species-specific in situ hybridisation probes. Individual pancreatic islets were obtained by laser-capture microdissection and subjected to analysis of mRNA expression of VMAT2. Results The VMAT2 protein was not expressed in beta cells in the adult pancreas of common mouse or rat laboratory strains, in contrast to its expression in beta cells (but not other pancreatic endocrine cell types) in the pancreas of pigs and humans. VMAT2- and tyrosine hydroxylase co-positive (catecholaminergic) innervation was less abundant in humans than in rodents. VMAT2-positive mast cells were identified in the pancreas of all species. Conclusions/interpretation Primates and pigs are suitable models for TBZ imaging of beta cells. Rodents, because of a complete lack of VMAT2 expression in the endocrine pancreas, are a ‘null’ model for assessing interference with BCM measurements by VMAT2-positive mast cells and sympathetic innervation in the pancreas. PMID:23404442

Development of a novel digestion chamber for human and porcine islet isolation.

PubMed

Gray, D W R; Sudhakaran, N; Titus, T T; McShane, P; Johnson, P

2004-05-01

The current technique of human pancreas digestion for islet isolation relies on selective distribution of collagenase delivered via the pancreatic duct to produce digestion and removal of peri-acinar fibrous tissue. However, the collagenase has relatively little effect on the interlobular fibrous tissue, which must therefore be broken down by mechanical means within the digestion chamber so as to release the contained acini and islets. The current way of achieving this in the Ricordi chamber is to place five or six stainless steel balls within the chamber and shake vigorously. The shaking presumably breaks down the interlobular fibrous tissue by a combination of shear force induced by the movement of tissue through the shaking process, assisted by numerous blows from the steel balls. Intuitively, one would expect some islets would be destroyed rather than released by such a battering. In an attempt to improve the efficiency of islet isolation we have designed a new digestion/filtration chamber that consists of a glass cylinder, sealed with Teflon plates holding in mesh filters at each end, secured in place by a central threaded tie-rod and external knurled nuts. A ring-shaped piston within the cylinder can be pushed up and down the travel by two rods passing out through sealed ports in the Teflon disk at one end and connected to an external handle. The handle is used to gently push the piston up and down the travel of the cylinder, which pushes the fluid and tissue through the central lumen of the ring-piston. A series of hooks attached to the central tie-rod catch the fibrous strands of the passing tissue; the shearing forces produced cause disruption by a process thought to be similar to teasing the tissue apart with fine forceps. A series of initial experiments with human pancreas showed the prototype to be too large, causing temperature control problems, and a redesigned smaller chamber was produced, maintaining the crucial design features. Experience processing five human pancreata has now demonstrated that in three of five pancreata the new chamber produced a good yield (>200,000 I.E.) of remarkably well separated and intact islets, the entire dispersion process being under 1 hour. However, in two isolations the collagenase digestion was poor, with few free islets. A copy of the new chamber (reserved for porcine work only) has been produced, as well as a copy of the Ricordi chamber. We have confirmed that the new chamber can isolate porcine islets in large numbers (>5000 islets/g pancreas [n = 2], but note that pig islets are small). These preliminary studies are sufficiently encouraging to justify further direct comparison with the Ricordi chamber for the purpose of animal and human islet isolation.

Magnetic resonance imaging: A tool to monitor and optimize enzyme distribution during porcine pancreas distention for islet isolation

PubMed Central

Scott, WE; Weegman, BP; Balamurugan, AN; Ferrer-Fabrega, J; Anazawa, T; Karatzas, T; Jie, T; Hammer, BE; Matsumoto, S; Avgoustiniatos, ES; Maynard, KS; Sutherland, DER; Hering, BJ; Papas, KK

2014-01-01

Background Porcine islet xenotransplantation is emerging as a potential alternative for allogeneic clinical islet transplantation. Optimization of porcine islet isolation in terms of yield and quality is critical for the success and cost effectiveness of this approach. Incomplete pancreas distension and inhomogeneous enzyme distribution have been identified as key factors for limiting viable islet yield per porcine pancreas. The aim of this study was to explore the utility of Magnetic Resonance Imaging (MRI) as a tool to investigate the homogeneity of enzyme delivery in porcine pancreata. Traditional and novel methods for enzyme delivery aimed at optimizing enzyme distribution were examined. Methods Pancreata were procured from Landrace pigs via en bloc viscerectomy. The main pancreatic duct was then cannulated with an 18g winged catheter and MRI performed at 1.5 T. Images were collected before and after ductal infusion of chilled MRI contrast agent (gadolinium) in physiological saline. Results Regions of the distal aspect of the splenic lobe and portions of the connecting lobe and bridge exhibited reduced delivery of solution when traditional methods of distension were utilized. Use of alternative methods of delivery (such as selective re-cannulation and distension of identified problem regions) resolved these issues and MRI was successfully utilized as a guide and assessment tool for improved delivery. Conclusion Current methods of porcine pancreas distension do not consistently deliver enzyme uniformly or adequately to all regions of the pancreas. Novel methods of enzyme delivery should be investigated and implemented for improved enzyme distribution. MRI serves as a valuable tool to visualize and evaluate the efficacy of current and prospective methods of pancreas distension and enzyme delivery. PMID:24986758

Magnetic resonance imaging: a tool to monitor and optimize enzyme distribution during porcine pancreas distention for islet isolation.

PubMed

Scott, William E; Weegman, Bradley P; Balamurugan, Appakalai N; Ferrer-Fabrega, Joana; Anazawa, Takayuki; Karatzas, Theodore; Jie, Tun; Hammer, Bruce E; Matsumoto, Shuchiro; Avgoustiniatos, Efstathios S; Maynard, Kristen S; Sutherland, David E R; Hering, Bernhard J; Papas, Klearchos K

2014-01-01

Porcine islet xenotransplantation is emerging as a potential alternative for allogeneic clinical islet transplantation. Optimization of porcine islet isolation in terms of yield and quality is critical for the success and cost-effectiveness of this approach. Incomplete pancreas distention and inhomogeneous enzyme distribution have been identified as key factors for limiting viable islet yield per porcine pancreas. The aim of this study was to explore the utility of magnetic resonance imaging (MRI) as a tool to investigate the homogeneity of enzyme delivery in porcine pancreata. Traditional and novel methods for enzyme delivery aimed at optimizing enzyme distribution were examined. Pancreata were procured from Landrace pigs via en bloc viscerectomy. The main pancreatic duct was then cannulated with an 18-g winged catheter and MRI performed at 1.5-T. Images were collected before and after ductal infusion of chilled MRI contrast agent (gadolinium) in physiological saline. Regions of the distal aspect of the splenic lobe and portions of the connecting lobe and bridge exhibited reduced delivery of solution when traditional methods of distention were utilized. Use of alternative methods of delivery (such as selective re-cannulation and distention of identified problem regions) resolved these issues, and MRI was successfully utilized as a guide and assessment tool for improved delivery. Current methods of porcine pancreas distention do not consistently deliver enzyme uniformly or adequately to all regions of the pancreas. Novel methods of enzyme delivery should be investigated and implemented for improved enzyme distribution. MRI serves as a valuable tool to visualize and evaluate the efficacy of current and prospective methods of pancreas distention and enzyme delivery. © 2014 John Wiley & Sons A/S Published by John Wiley & Sons Ltd.

Adjuvant effect of Gantrez®AN nanoparticles during oral vaccination of piglets against F4+enterotoxigenic Escherichia coli.

PubMed

Vandamme, Katrien; Melkebeek, Vesna; Vesna, Melkebeek; Cox, Eric; Eric, Cox; Remon, Jean Paul; Paul, Remon Jean; Vervaet, Chris; Chris, Vervaet

2011-02-15

In this study, the adjuvanticity of methylvinylether-co-maleic anhydride (Gantrez(®)AN) nanoparticles (NP) was investigated in an oral immunisation experiment of pigs against F4+enterotoxigenic Escherichia coli (F4+ETEC). In addition, Wheat Germ Agglutinin (WGA)-coating of the nanoparticles was tested for enterocyte-targeting. Pigs were either vaccinated with F4 fimbriae, F4 encapsulated in Gantrez(®)AN NP, F4 encapsulated in Gantrez(®)AN NP coated with WGA or F4 fimbriae mixed with empty Gantrez(®)AN NP. Only vaccination with the combination of F4 mixed with empty Gantrez(®)AN NP improved protection against F4+ETEC infection. In addition, vaccination with this formulation also resulted in an F4-specific serum antibody response prior to F4+ETEC challenge. Encapsulation of F4 in Gantrez(®)AN NP only raised the serum antibody response after F4+ETEC challenge compared to soluble F4, but did not improve protection, whereas WGA-coating almost completely abolished the serum antibody response. These data indicate that nanoparticle effects after F4 encapsulation were of lesser importance for the adjuvant effect of Gantrez(®)AN NP, contrarily to the reactivity of the Gantrez(®)AN polymer used to prepare the nanoparticles. Copyright © 2010 Elsevier B.V. All rights reserved.

Ubiquitous LEA29Y Expression Blocks T Cell Co-Stimulation but Permits Sexual Reproduction in Genetically Modified Pigs.

PubMed

Bähr, Andrea; Käser, Tobias; Kemter, Elisabeth; Gerner, Wilhelm; Kurome, Mayuko; Baars, Wiebke; Herbach, Nadja; Witter, Kirsti; Wünsch, Annegret; Talker, Stephanie C; Kessler, Barbara; Nagashima, Hiroshi; Saalmüller, Armin; Schwinzer, Reinhard; Wolf, Eckhard; Klymiuk, Nikolai

2016-01-01

We have successfully established and characterized a genetically modified pig line with ubiquitous expression of LEA29Y, a human CTLA4-Ig derivate. LEA29Y binds human B7.1/CD80 and B7.2/CD86 with high affinity and is thus a potent inhibitor of T cell co-stimulation via this pathway. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via assisted breeding methods. The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level. They present with an immune system affected by transgene expression, but can be maintained until sexual maturity and propagated by assisted reproduction techniques. Based on previous experience with pancreatic islets expressing LEA29Y, tissues from CAG-LEA29Y transgenic pigs should be protected against rejection by human T cells. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting large animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action.

CRYOPRESERVATION EFFECTS ON RECOMBINANT MYOBLASTS ENCAPSULATED IN ADHESIVE ALGINATE HYDROGELS

PubMed Central

Ahmad, Hajira F.; Sambanis, Athanassios

2013-01-01

Cell encapsulation in hydrogels is widely used in tissue engineering applications, including encapsulation of islets or other insulin-secreting cells in pancreatic substitutes. Use of adhesive, bio-functionalized hydrogels is receiving increasing attention, as cell-matrix interactions in 3-D can be important for various cell processes. With pancreatic substitutes, studies have indicated benefits of 3-D adhesion on the viability and/or function of insulin-secreting cells. As long-term storage of microencapsulated cells is critical for their clinical translation, cryopreservation of cells in hydrogels is actively being investigated. Previous studies have examined the cryopreservation response of cells encapsulated in non-adhesive hydrogels using conventional freezing and/or vitrification (ice-free cryopreservation), however, none have systematically compared the two cryopreservation methods with cells encapsulated within an adhesive 3-D environment. The latter would be significant, as evidence suggests adhesion influences cellular response to cryopreservation. Thus, the objective of this study was to determine the response to conventional freezing and vitrification of insulin-secreting cells encapsulated in an adhesive biomimetic hydrogel. Recombinant insulin-secreting C2C12 myoblasts were encapsulated in oxidized RGD-alginate and cultured 1 or 4 days post-encapsulation, cryopreserved, and assessed up to 3 days post-warming for metabolic activity and insulin secretion, and one day post-warming for cell morphology. Besides certain transient differences of the vitrified group relative to the Fresh control, both conventional freezing and vitrification maintained metabolism, secretion and morphology of the recombinant C2C12 cells. Thus, due to a simpler procedure and slightly superior results, conventional freezing is recommended over vitrification for the cryopreservation of C2C12 cells in oxidized RGD-modified alginate. PMID:23499987

Hydrogel-based encapsulation of biological, functional tissue: fundamentals, technologies and applications

NASA Astrophysics Data System (ADS)

Zimmermann, H.; Ehrhart, F.; Zimmermann, D.; Müller, K.; Katsen-Globa, A.; Behringer, M.; Feilen, P. J.; Gessner, P.; Zimmermann, G.; Shirley, S. G.; Weber, M. M.; Metze, J.; Zimmermann, U.

2007-12-01

Replacing dysfunctional endocrine cells or tissues (e.g. islets, parathyroid tissue) by functional, foreign material without using immunosuppressives could soon become reality. Immunological reactions are avoided by encapsulating cells/tissues in hydrogel (e.g. alginate) microcapsules, preventing interaction of the enclosed material with the host’s immune system while permitting the unhindered passage of nutrients, oxygen and secreted therapeutic factors. Detailed investigations of the physical, physico-chemical and immunological parameters of alginate-based microcapsules have led recently to the development of a novel class of cell-entrapping microcapsules that meet the demands of biocompatibility, long-term integrity and function. This together with the development of ‘good medical practice’ microfluidic chip technology and of advanced cryopreservation technology for generation and storage of immunoisolated transplants will bring cell-based therapy to clinics and the market.

Topical delivery of liposomally encapsulated interferon evaluated in a cutaneous herpes guinea pig model.

PubMed Central

Weiner, N; Williams, N; Birch, G; Ramachandran, C; Shipman, C; Flynn, G

1989-01-01

The topical delivery of liposomally encapsulated interferon was evaluated in the cutaneous herpes simplex virus guinea pig model. Application of liposomally entrapped interferon caused a reduction of lesion scores, whereas application of interferon formulated as a solution or as an emulsion was ineffective. The method of liposomal preparation rather than the lipid composition of the bilayers appeared to be the most important factor for reducing lesion scores. Only liposomes prepared by the dehydration-rehydration method were effective. This finding implied that the dehydration and subsequent rehydration of the liposomes facilitate partitioning of the interferon into liposomal bilayers, where the drug is positioned for transfer into the lipid compartment of the stratum corneum. Liposomes do not appear to function as permeation enhancers but seem to provide the needed physicochemical environment for transfer of interferon into the skin. PMID:2802550

Attachment of alginate microcapsules onto plasma-treated PDMS sheet for retrieval after transplantation.

PubMed

Shin, Soojeong; Shin, Jeong Eun; Yoo, Young Je

2013-01-01

Although transplantation of microencapsulated islets has been proposed as a therapy for the treatment of diabetes mellitus, limited retrievability of the cells has impeded its medical usage. To achieve retrieval of microencapsulated islets, capsules were attached to polydimethylsiloxane (PDMS) with a biocompatible adhesive. Because the hydrophobic nature of the PDMS surface prevents attachment, surface modification is essential. Alginate microcapsules were attached to modified PDMS sheets, and the mechanical stability of the resulting constructs was determined. Acrylic acid (AA) and acrylamide (AM) mixtures were grafted on the surfaces of PDMS sheets using a two-step oxygen plasma treatment (TSPT). TSPT-PDMS was characterized according to water contact angle and zeta-potential measurements. The contact angle was altered by changing the ratio of AM to AA to generate hydrophilic surface. Evaluation of the surface charge at pH 2, 7, and 12 confirmed the presence of polar groups on the modified surface. Microcapsules were attached to TSPT-PDMS using Histoacryl® and shown to be in a monolayered and half-exposed state. The shear stress resistance of alginate capsules attached to the PDMS sheet indicates the possibility of transplantation of encapsulated cells without scattering in vivo. This method is applicable to retrieve microencapsulated porcine islets when required. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

Evans Blue Staining Reveals Vascular Leakage Associated with Focal Areas of Host-Parasite Interaction in Brains of Pigs Infected with Taenia solium

PubMed Central

Paredes, Adriana; Cangalaya, Carla; Rivera, Andrea; Gonzalez, Armando E.; Mahanty, Siddhartha; Garcia, Hector H.; Nash, Theodore E.

2014-01-01

Cysticidal drug treatment of viable Taenia solium brain parenchymal cysts leads to an acute pericystic host inflammatory response and blood brain barrier breakdown (BBB), commonly resulting in seizures. Naturally infected pigs, untreated or treated one time with praziquantel were sacrificed at 48 hr and 120 hr following the injection of Evans blue (EB) to assess the effect of treatment on larval parasites and surrounding tissue. Examination of harvested non encapsulated muscle cysts unexpectedly revealed one or more small, focal round region(s) of Evans blue dye infiltration (REBI) on the surface of otherwise non dye-stained muscle cysts. Histopathological analysis of REBI revealed focal areas of eosinophil-rich inflammatory infiltrates that migrated from the capsule into the tegument and internal structures of the parasite. In addition some encapsulated brain cysts, in which the presence of REBI could not be directly assessed, showed histopathology identical to that of the REBI. Muscle cysts with REBI were more frequent in pigs that had received praziquantel (6.6% of 3736 cysts; n = 6 pigs) than in those that were untreated (0.2% of 3172 cysts; n = 2 pigs). Similar results were found in the brain, where 20.7% of 29 cysts showed histopathology identical to muscle REBI cysts in praziquantel-treated pigs compared to the 4.3% of 47 cysts in untreated pigs. Closer examination of REBI infiltrates showed that EB was taken up only by eosinophils, a major component of the cellular infiltrates, which likely explains persistence of EB in the REBI. REBI likely represent early damaging host responses to T. solium cysts and highlight the focal nature of this initial host response and the importance of eosinophils at sites of host-parasite interaction. These findings suggest new avenues for immunomodulation to reduce inflammatory side effects of anthelmintic therapy. PMID:24915533

Manufacturing porcine islets: culture at 22 °C has no advantage above culture at 37 °C: a gene expression evaluation.

PubMed

Mueller, Kate R; Martins, Kyra V; Murtaugh, Michael P; Schuurman, Henk-Jan; Papas, Klearchos K

2013-01-01

The manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs. Porcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT-PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers. Post-culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129-159 nmol/min·mgDNA before culture, and 259-291, 204-212, and 207-228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10-fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re-equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors. Culture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress. © 2013 John Wiley & Sons A/S.

Islet amyloid polypeptide (IAPP):cDNA cloning and identification of an amyloidogenic region associated with the species-specific occurrence of age-related diabetes mellitus.

PubMed

Betsholtz, C; Svensson, V; Rorsman, F; Engström, U; Westermark, G T; Wilander, E; Johnson, K; Westermark, P

1989-08-01

We have cloned and sequenced a human islet amyloid polypeptide (IAPP) cDNA. A secretory 89 amino acid IAPP protein precursor is predicted from which the 37 amino acid IAPP molecule is formed by amino- and carboxyterminal proteolytic processing. The IAPP peptide is 43-46% identical in amino acid sequence to the two members of the calcitonin gene-related peptide (CGRP) family. Evolutionary conserved proteolytic processing sites indicate that similar proteases are involved in the maturation of IAPP and CGRP and that the IAPP amyloid polypeptide is identical to the normal proteolytic product of the IAPP precursor. A synthetic peptide corresponding to a carboxyteminal fragment of human IAPP is shown to spontaneously form amyloid-like fibrils in vitro. Antibodies against this peptide cross-react with IAPP from species that develop amyloid in pancreatic islets in conjunction with age-related diabetes mellitus (human, cat, racoon), but do not cross-react with IAPP from other tested species (mouse, rat, guinea pig, dog). Thus, a species-specific structural motif in the putative amyloidogenic region of IAPP is associated with both amyloid formation and the development of age-related diabetes mellitus. This provides a new molecular clue to the pathogenesis of this disease.

Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system.

PubMed

Cho, Bumrae; Kim, Su Jin; Lee, Eun-Jin; Ahn, Sun Mi; Lee, Jin Seok; Ji, Dal-Young; Lee, Kiho; Kang, Jung-Taek

2018-06-01

Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.

Transgenic-cloned pigs systemically expressing red fluorescent protein, Kusabira-Orange.

PubMed

Matsunari, Hitomi; Onodera, Masafumi; Tada, Norihiro; Mochizuki, Hideki; Karasawa, Satoshi; Haruyama, Erika; Nakayama, Naoki; Saito, Hitoshi; Ueno, Satoshi; Kurome, Mayuko; Miyawaki, Atsushi; Nagashima, Hiroshi

2008-09-01

Genetically engineered pigs with cell markers such as fluorescent proteins are highly useful in lines of research that include the tracking of transplanted cells or tissues. In this study, we produced transgenic-cloned pigs carrying a gene for the newly developed red fluorescent protein, humanized Kusabira-Orange (huKO), which was cloned from the coral stone Fungia concinna. The nuclear transfer embryos, reconstructed with fetal fibroblast cells that had been transduced with huKO cDNA using retroviral vector D Delta Nsap, developed efficiently in vitro into blastocysts (28.0%, 37/132). Nearly all (94.6%, 35/37) of the cloned blastocysts derived from the transduced cells exhibited clear huKO gene expression. A total of 429 nuclear transfer embryos were transferred to four recipients, all of which became pregnant and gave birth to 18 transgenic-cloned offspring in total. All of the pigs highly expressed huKO fluorescence in all of the 23 organs and tissues analyzed, including the brain, eyes, intestinal and reproductive organs, skeletal muscle, bone, skin, and hoof. Furthermore, such expression was also confirmed by histological analyses of various tissues such as pancreatic islets, renal corpuscles, neuronal and glial cells, the retina, chondrocytes, and hematopoietic cells. These data demonstrate that transgenic-cloned pigs exhibiting systemic red fluorescence expression can be efficiently produced by nuclear transfer of somatic cells retrovirally transduced with huKO gene.

Extended Microbiological Characterization of Göttingen Minipigs in the Context of Xenotransplantation: Detection and Vertical Transmission of Hepatitis E Virus

PubMed Central

Morozov, Vladimir A.; Morozov, Alexey V.; Rotem, Avi; Barkai, Uriel; Bornstein, Stefan; Denner, Joachim

2015-01-01

Xenotransplantation has been proposed as a solution to the shortage of suitable human donors. Pigs are currently favoured as donor animals for xenotransplantation of cells, including islet cells, or organs. To reduce the xenotransplantation-associated risk of infection of the recipient the pig donor should be carefully characterised. Göttingen minipigs from Ellegaard are often used for biomedical research and are regularly tested by their vendor for the presence of numerous bacteria, fungi, viruses and parasites. However, screening for some pathogens transmittable to humans had not been performed.The presence of microorganisms was examined in Göttingen Minipigs by PCR methods. Since zoonotic transmission of porcine hepatitis E virus HEV to humans has been demonstrated, extended search for HEV was considered as a priority. RNA from sera, islet and other cells from 40 minipigs were examined for HEV using different real-time reverse transcription (RT)-PCRs, among them two newly established. In addition, sera were examined by Western blot analysis using two recombinant capsid proteins of HEV as antigens. HEV RNA was not detected in pigs older than one year including gilts, but it was detected in the sera of three of ten animals younger than 1 year. Furthermore, HEV was also detected in the sera of three sows six days after delivery and their offspring, indicating vertical transmission of the virus. PCR amplicons were cloned, sequenced and the viruses were found to belong to the HEV genotype (gt) 3/4. Anti-HEV immunoglobulins G were detected in one sow and maternal antibodies in her six day old piglet. Since Göttingen minipigs were negative for many xenotransplantation-relevant microorganisms, they can now be classified as safe. HEV may be eliminated from the Ellegaard herd by selection of negative animals and/or by treatment of the animals. PMID:26466154

Microencapsulation of islets within alginate/poly(ethylene glycol) gels cross-linked via Staudinger ligation

PubMed Central

Hall, Kristina K.; Gattás-Asfura, Kerim M.; Stabler, Cherie L.

2010-01-01

Functionalized alginate and PEG polymers were used to generate covalently linked alginate-PEG (XAlgPEG) microbeads of high stability. The cell-compatible Staudinger ligation scheme was used to chemoselectively cross-link phosphine-terminated poly(ethylene glycol) (PEG) to azide-functionalized alginate, resulting in XAlgPEG hydrogels. XAlgPEG microbeads were formed by co-incubation of the two polymers, followed by ionic cross-linking of the alginate using barium ions. The enhanced stability and gel properties of the resulting XAlgPEG microbeads, as well as the compatibility of these polymers for the encapsulation of islets and beta cells lines, were investigated. Our data show that XAlgPEG microbeads exhibit superior resistance to osmotic swelling compared to traditional barium cross-linked alginate (Ba-Alg) beads, with a 5-fold reduction in observed swelling, as well as resistance to dissolution via chelation solution. Diffusion and porosity studies found XAlgPEG beads to exhibit properties comparable to standard Ba-Alg. Our data found XAlgPEG microbeads to be highly cell compatible with insulinoma cell lines, as well as rat and human pancreatic islets, where the viability and functional assessment of cells within XAlgPEG were comparable to Ba-Alg controls. The remarkable improved stability, as well as demonstrated cellular compatibility, of XAlgPEG hydrogels makes them an appealing option for a wide variety of tissue engineering applications. PMID:20654745

Sustained Pulmonary Delivery of a Water-Soluble Antibiotic Without Encapsulating Carriers.

PubMed

Ong, Winston; Nowak, Pawel; Cu, Yen; Schopf, Lisa; Bourassa, James; Enlow, Elizabeth; Moskowitz, Samuel M; Chen, Hongming

2016-03-01

Traditional polymeric nanoparticle formulations for prolonged local action during inhalation therapy are highly susceptible to muco-ciliary clearance. In addition, polymeric carriers are typically administered in high doses due to finite drug loading. For toxicological reasons, these carriers and their degradation byproducts are undesirable for inhalation therapy, particularly for chronic use, due to potential lung accumulation. We synthesized a novel, insoluble prodrug (MRPD) of a time-dependent β-lactam, meropenem, and formulated MRPD into mucus-penetrating crystals (MRPD-MPCs). After characterizing their mucus mobility (in vitro) and stability, we evaluated the lung pharmacokinetics of intratracheally-instilled MRPD-MPCs and a meropenem solution in guinea pigs. Meropenem levels rapidly declined in the lungs of guinea pigs receiving meropenem solution compared to those given MRPD-MPCs. At 9 h after dosing, drug levels in the lungs of animals that received meropenem solution dropped to 12 ng/mL, whereas those that received MRPD-MPCs maintained an average drug level of ≥1,065 ng/mL over a 12-h period. This work demonstrated that the combination of prodrug chemistry and mucus-penetrating platform created nanoparticles that produced sustained levels of meropenem in guinea pig lungs. This strategy represents a novel approach for sustained local drug delivery to the lung without using encapsulating matrices.

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

PubMed Central

Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J.; Kim, Hyunil; Surh, Charles D.; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation. PMID:23071605

Generation and characterization of human heme oxygenase-1 transgenic pigs.

PubMed

Yeom, Hye-Jung; Koo, Ok Jae; Yang, Jaeseok; Cho, Bumrae; Hwang, Jong-Ik; Park, Sol Ji; Hurh, Sunghoon; Kim, Hwajung; Lee, Eun Mi; Ro, Han; Kang, Jung Taek; Kim, Su Jin; Won, Jae-Kyung; O'Connell, Philip J; Kim, Hyunil; Surh, Charles D; Lee, Byeong-Chun; Ahn, Curie

2012-01-01

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Pig islet xenotransplantation acceptance in a Latin-American diabetic population.

PubMed

Abalovich, Adrián; Wechsler, Carlos; Lara, Silvia; Bervottini, Miguel

2010-01-01

Progress in porcine islet xenotransplantation has been accompanied by studies on acceptance of this new procedure by patients, health professionals or the general public. Such studies have not been done in the Latin-American population. We conducted a questionnaire in 108 diabetes patients (insulin-dependent, n = 53; insulin-independent, n = 55) in a public hospital in Argentina. The questions addressed the general perception of the xenotransplant procedure and specific items related to the outcome (achieving insulin independence, improvement in metabolic control, delay in emergence of diabetic complications, need for repeat procedures, potential of transfer of infectious viruses, association with psychological problems, and anticipated success in relation to achieving a cure). Eighty-six (79%) of the patients accepted islet xenotransplantation; this incidence was not different for insulin-dependent or insulin-independent patients, patients with or without complications, or patients with good or poor metabolic control. Also, over 75% of patients accepted the procedure if this is only associated with a reduction in insulin requirement, if the procedure just delays but not prevents the onset of complications, or if the procedure needs to be performed every 6 months. Fifty-seven percent of patients indicated acceptance even if the potential transmission of a virus infection cannot be completely ruled out: this outcome was not affected by the outbreak of the H1N1 flu epidemic during the conduct of this study. Forty percent of patients indicated that living with porcine cells in their body could give psychological problems. We conclude that this population of Latin-American diabetic patients shows a high acceptance rate of a porcine islet xenotransplantation product. (c) 2010 John Wiley & Sons A/S.

Assessment of Immune Isolation of Allogeneic Mouse Pancreatic Progenitor Cells by a Macroencapsulation Device

PubMed Central

Faleo, Gaetano; Lee, Karim; Nguyen, Vinh; Tang, Qizhi

2016-01-01

Background Embryonic-stem-cell (ESC)-derived islets hold the promise of providing a renewable source of tissue for the treatment of insulin-dependent diabetes. Encapsulation may allow ESC-derived islets to be transplanted without immunosuppression, thus enabling wider application of this therapy. Methods In this study, we investigated the immunogenicity of mouse pancreatic progenitor cells and efficacy of a new macroencapsulation device in protecting these cells against alloimmune and autoimmune responses in mouse models. Results Mouse pancreatic progenitor cells activated the indirect but not the direct pathway of alloimmune response and were promptly rejected in immune competent hosts. The new macroencapsulation device abolished T cell activation induced by allogeneic splenocytes and protected allogeneic MIN6 β cells and pancreatic progenitors from rejection even in pre-sensitized recipients. In addition, the device was effective in protecting MIN6 cells in spontaneously diabetic non-obese diabetic recipients against both alloimmune and recurring autoimmune responses. Conclusion Our results demonstrate that macroencapsulation can effectively prevent immune sensing and rejection of allogeneic pancreatic progenitor cells in fully sensitized and autoimmune hosts. PMID:26982952

Mucosal Immunity and Protective Efficacy of Intranasal Inactivated Influenza Vaccine Is Improved by Chitosan Nanoparticle Delivery in Pigs.

PubMed

Dhakal, Santosh; Renu, Sankar; Ghimire, Shristi; Shaan Lakshmanappa, Yashavanth; Hogshead, Bradley T; Feliciano-Ruiz, Ninoshkaly; Lu, Fangjia; HogenEsch, Harm; Krakowka, Steven; Lee, Chang Won; Renukaradhya, Gourapura J

2018-01-01

Annually, swine influenza A virus (SwIAV) causes severe economic loss to swine industry. Currently used inactivated SwIAV vaccines administered by intramuscular injection provide homologous protection, but limited heterologous protection against constantly evolving field viruses, attributable to the induction of inadequate levels of mucosal IgA and cellular immune responses in the respiratory tract. A novel vaccine delivery platform using mucoadhesive chitosan nanoparticles (CNPs) administered through intranasal (IN) route has the potential to elicit strong mucosal and systemic immune responses in pigs. In this study, we evaluated the immune responses and cross-protective efficacy of IN chitosan encapsulated inactivated SwIAV vaccine in pigs. Killed SwIAV H1N2 (δ-lineage) antigens (KAg) were encapsulated in chitosan polymer-based nanoparticles (CNPs-KAg). The candidate vaccine was administered twice IN as mist to nursery pigs. Vaccinates and controls were then challenged with a zoonotic and virulent heterologous SwIAV H1N1 (γ-lineage). Pigs vaccinated with CNPs-KAg exhibited an enhanced IgG serum antibody and mucosal secretory IgA antibody responses in nasal swabs, bronchoalveolar lavage (BAL) fluids, and lung lysates that were reactive against homologous (H1N2), heterologous (H1N1), and heterosubtypic (H3N2) influenza A virus strains. Prior to challenge, an increased frequency of cytotoxic T lymphocytes, antigen-specific lymphocyte proliferation, and recall IFN-γ secretion by restimulated peripheral blood mononuclear cells in CNPs-KAg compared to control KAg vaccinates were observed. In CNPs-KAg vaccinated pigs challenged with heterologous virus reduced severity of macroscopic and microscopic influenza-associated pulmonary lesions were observed. Importantly, the infectious SwIAV titers in nasal swabs [days post-challenge (DPC) 4] and BAL fluid (DPC 6) were significantly ( p  < 0.05) reduced in CNPs-KAg vaccinates but not in KAg vaccinates when compared to the unvaccinated challenge controls. As well, an increased frequency of T helper memory cells and increased levels of recall IFNγ secretion by tracheobronchial lymph nodes cells were observed. In summary, chitosan SwIAV nanovaccine delivered by IN route elicited strong cross-reactive mucosal IgA and cellular immune responses in the respiratory tract that resulted in a reduced nasal viral shedding and lung virus titers in pigs. Thus, chitosan-based influenza nanovaccine may be an ideal candidate vaccine for use in pigs, and pig is a useful animal model for preclinical testing of particulate IN human influenza vaccines.

Towards retrievable vascularized bioartificial pancreas: induction and long-lasting stability of polymeric mesh implant vascularized with the help of acidic and basic fibroblast growth factors and hydrogel coating.

PubMed

Prokop, A; Kozlov, E; Nun Non, S; Dikov, M M; Sephel, G C; Whitsitt, J S; Davidson, J M

2001-01-01

We seek to improve existing methodologies for allogenic grafting of pancreatic islets. The lack of success of encapsulated transplanted islets inside the peritoneal cavity is presently attributed to poor vascularization of the implant. A thick, fibrotic capsule often surrounds the graft, limiting survival. We have tested the hypothesis that neovascularization of the graft material can be induced by the addition of proper angiogenic factors embedded within a polymeric coat. Biocompatible and nonresorbable meshes coated with hydrophilic polymers were implanted in rats and harvested after 1-, 6-, and 12-week intervals. The implant response was assessed by histological observations on the degree of vascularity, fibrosis, and inflammation. Macrostructural geometry of meshes was conducive to tissue ingrowth into the interstitial space between the mesh filaments. Hydrogel coating with incorporated acidic or basic FGF in an electrostatic complex with polyelectrolytes and/or with heparin provided a sustained slow release of the angiogenic growth factor. Anti-factor VIII and anti-collagen type IV antibodies and a GSL I-B4 lectin were used to measure the extent of vascularization. Vigorous and persistent vascularization radiated several hundred microns from the implant. The level of vascularization should provide a sufficient diffusion of nutrients and oxygen to implanted islets. Based on our observations, stable vascularization may require a sustained angiogenic signal to allow for the development of a permanent implant structure.

Preventing hypoxia-induced cell death in beta cells and islets via hydrolytically activated, oxygen-generating biomaterials

PubMed Central

Pedraza, Eileen; Coronel, Maria M.; Fraker, Christopher A.; Ricordi, Camillo; Stabler, Cherie L.

2012-01-01

A major hindrance in engineering tissues containing highly metabolically active cells is the insufficient oxygenation of these implants, which results in dying or dysfunctional cells in portions of the graft. The development of methods to increase oxygen availability within tissue-engineered implants, particularly during the early engraftment period, would serve to allay hypoxia-induced cell death. Herein, we designed and developed a hydrolytically activated oxygen-generating biomaterial in the form of polydimethylsiloxane (PDMS)-encapsulated solid calcium peroxide, PDMS-CaO2. Encapsulation of solid peroxide within hydrophobic PDMS resulted in sustained oxygen generation, whereby a single disk generated oxygen for more than 6 wk at an average rate of 0.026 mM per day. The ability of this oxygen-generating material to support cell survival was evaluated using a β cell line and pancreatic rat islets. The presence of a single PDMS-CaO2 disk eliminated hypoxia-induced cell dysfunction and death for both cell types, resulting in metabolic function and glucose-dependent insulin secretion comparable to that in normoxic controls. A single PDMS-CaO2 disk also sustained enhanced β cell proliferation for more than 3 wk under hypoxic culture conditions. Incorporation of these materials within 3D constructs illustrated the benefits of these materials to prevent the development of detrimental oxygen gradients within large implants. Mathematical simulations permitted accurate prediction of oxygen gradients within 3D constructs and highlighted conditions under which supplementation of oxygen tension would serve to benefit cellular viability. Given the generality of this platform, the translation of these materials to other cell-based implants, as well as ischemic tissues in general, is envisioned. PMID:22371586

Niosomal encapsulation of ethambutol hydrochloride for increasing its efficacy and safety.

PubMed

El-Ridy, Mohammed Shafik; Yehia, Soad Aly; Kassem, Mahfouz Abd-El-Megeid; Mostafa, Dina Mahmoud; Nasr, Essam Amin; Asfour, Marwa Hasanin

2015-01-01

Tuberculosis (TB) is a worldwide health concern. In 2011, about 8.7 million new cases developed TB and 1.4 million people died from it. Enhancement of ethambutol hydrochloride activity and safety in treatment of TB through niosomal encapsulation. Niosomes were prepared by the thin-film hydration method. They were characterized, investigated for in vitro release, lung disposition and in vivo biological evaluation. Entrapment efficiency of ethambutol hydrochloride ranged from 12.20% to 25.81%. Zeta potential values inferred stability of neutral and negatively charged formulations. In vitro release was biphasic. Lung targeting was increased by niosomal encapsulation. Biological evaluation revealed superiority of niosomal ethambutol hydrochloride over the free drug. Neutral and negatively charged niosomal vesicles are dispersed homogenously unlike positively charged vesicles. Niosomal encapsulation results in controlled drug release. Niosomal formulations targeted more drugs to mice lungs for a prolonged period of time resulting in: decreased root-specific lung weight, bacterial counts in lung homogenates and optimizing pathological effect on guinea pigs lungs, livers and spleens. Encapsulation of ethambutol hydrochloride in niosomal formulations for the treatment of TB provides higher efficacy and safety compared with the free drug.

Histology, immunohistochemistry, and in situ hybridization reveal overlooked Ebola virus target tissues in the Ebola virus disease guinea pig model.

PubMed

Cooper, Timothy K; Huzella, Louis; Johnson, Joshua C; Rojas, Oscar; Yellayi, Sri; Sun, Mei G; Bavari, Sina; Bonilla, Amanda; Hart, Randy; Jahrling, Peter B; Kuhn, Jens H; Zeng, Xiankun

2018-01-19

Survivors of Ebola virus infection may become subclinically infected, but whether animal models recapitulate this complication is unclear. Using histology in combination with immunohistochemistry and in situ hybridization in a retrospective review of a guinea pig confirmation-of-virulence study, we demonstrate for the first time Ebola virus infection in hepatic oval cells, the endocardium and stroma of the atrioventricular valves and chordae tendinae, satellite cells of peripheral ganglia, neurofibroblasts and Schwann cells of peripheral nerves and ganglia, smooth muscle cells of the uterine myometrium and vaginal wall, acini of the parotid salivary glands, thyroid follicular cells, adrenal medullary cells, pancreatic islet cells, endometrial glandular and surface epithelium, and the epithelium of the vagina, penis and, prepuce. These findings indicate that standard animal models for Ebola virus disease are not as well-described as previously thought and may serve as a stepping stone for future identification of potential sites of virus persistence.

TRANSPLANTATION OF HEPATOCYTES FROM GENETICALLY-ENGINEERED PIGS IN BABOONS

PubMed Central

Iwase, Hayato; Liu, Hong; Schmelzer, Eva; Ezzelarab, Mohamed; Wijkstrom, Martin; Hara, Hidetaka; Lee, Whayoung; Singh, Jagjit; Long, Cassandra; Lagasse, Eric; Gerlach, Jörg C.; Cooper, David K.C.; Gridelli, Bruno

2017-01-01

Background Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically-engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. Methods Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by measurement of anti-nonGal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. Results Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low – 500–1,000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5,000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. Discussion and Conclusions As a result of this disappointing experience, the following points need to be considered. (i) Were the isolated pig hepatocytes functionally viable? (ii) Are pig hepatocytes more immunogenic than pig hearts, kidneys, artery patch grafts, or islets? (iii) Does injection of pig cells (antigens) into the spleen and/or lymph nodes stimulate a greater immune response than when pig tissues are grafted at other sites? (iv) Did the presence of the recipient’s intact liver prevent survival and proliferation of pig hepatocytes? (v) Is pig CD47-primate SIRP-α compatibility essential? In conclusion, the transplantation of genetically-engineered pig hepatocytes into multiple sites in immunosuppressed baboons was associated with very early graft failure. Considerable further study is required before clinical trials should be undertaken. PMID:28130881

Transplantation of hepatocytes from genetically engineered pigs into baboons.

PubMed

Iwase, Hayato; Liu, Hong; Schmelzer, Eva; Ezzelarab, Mohamed; Wijkstrom, Martin; Hara, Hidetaka; Lee, Whayoung; Singh, Jagjit; Long, Cassandra; Lagasse, Eric; Gerlach, Jörg C; Cooper, David K C; Gridelli, Bruno

2017-03-01

Some patients with acute or acute-on-chronic hepatic failure die before a suitable human liver allograft becomes available. Encouraging results have been achieved in such patients by the transplantation of human hepatocyte progenitor cells from fetal liver tissue. The aim of the study was to explore survival of hepatocytes from genetically engineered pigs after direct injection into the spleen and other selected sites in immunosuppressed baboons to monitor the immune response and the metabolic function and survival of the transplanted hepatocytes. Baboons (n=3) were recipients of GTKO/hCD46 pig hepatocytes. All three baboons received anti-thymocyte globulin (ATG) induction and tapering methylprednisolone. Baboon 1 received maintenance immunosuppressive therapy with tacrolimus and rapamycin. Baboons 2 and 3 received an anti-CD40mAb/rapamycin-based regimen that prevents sensitization to pig solid organ grafts. The baboons were euthanized 4 or 5 weeks after hepatocyte transplantation. The baboon immune response was monitored by the measurement of anti-non-Gal IgM and IgG antibodies (by flow cytometry) and CFSE-mixed lymphocyte reaction. Monitoring for hepatocyte survival and function was by (i) real-time PCR detection of porcine DNA, (ii) real-time PCR for porcine gene expression, and (iii) pig serum albumin levels (by ELISA). The sites of hepatocyte injection were examined microscopically. Detection of porcine DNA and porcine gene expression was minimal at all sites of hepatocyte injection. Serum levels of porcine albumen were very low-500-1000-fold lower than in baboons with orthotopic pig liver grafts, and approximately 5000-fold lower than in healthy pigs. No hepatocytes or infiltrating immune cells were seen at any of the injection sites. Two baboons (Baboons 1 and 3) demonstrated a significant increase in anti-pig IgM and an even greater increase in IgG, indicating sensitization to pig antigens. As a result of this disappointing experience, the following points need to be considered. (i) Were the isolated pig hepatocytes functionally viable? (ii) Are pig hepatocytes more immunogenic than pig hearts, kidneys, artery patch grafts, or islets? (iii) Does injection of pig cells (antigens) into the spleen and/or lymph nodes stimulate a greater immune response than when pig tissues are grafted at other sites? (iv) Did the presence of the recipient's intact liver prevent survival and proliferation of pig hepatocytes? (v) Is pig CD47-primate SIRP-α compatibility essential? In conclusion, the transplantation of genetically engineered pig hepatocytes into multiple sites in immunosuppressed baboons was associated with very early graft failure. Considerable further study is required before clinical trials should be undertaken. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Formulation and evaluation of lidocaine base ethosomes for transdermal delivery.

PubMed

Zhu, Xiaoliang; Li, Fuli; Peng, Xuebiao; Zeng, Kang

2013-08-01

Although transdermal preparations of local anesthetics have been used to reduce pain caused by skin surgery, these preparations cannot effectively penetrate through the epidermis because of the barrier formed by the stratum corneum and the thick epidermis. Ethosomes can effectively transport drugs across the skin because of their thermodynamic stability, small size, high encapsulation efficiency, and percutaneous penetration. We evaluated lidocaine base ethosomes by measuring their loading efficiency, encapsulation efficiency, thermodynamic stability, and percutaneous penetration capability in vitro, and their effectiveness and cutaneous irritation in vivo. Lidocaine base ethosomes were prepared using the injection-sonication-filter method. Size, loading efficiency, encapsulation efficiency, and stability were evaluated using a Zetasizer and high performance liquid chromatography. Formulation was determined by measuring the maximum encapsulation efficiency in the orthogonal test. Percutaneous penetration efficiency in vitro was analyzed using a Franz-type diffusion cell experiment. In vivo effectiveness was analyzed using the pinprick test. Cutaneous irritancy tests were performed on white guinea pigs, followed by histopathologic analysis. The results were compared with lidocaine liposomes as well as lidocaine delivered in a hydroethanolic solution. Lidocaine base ethosomes composed of 5% (w/w) egg phosphatidyl choline, 35% (w/w) ethanol, 0.2% (w/w) cholesterol, 5% (w/w) lidocaine base, and ultrapure water had a mean maximum encapsulation of 51% ± 4%, a mean particle size of 31 ± 3 nm, and a mean loading efficiency of 95.0% ± 0.1%. The encapsulation efficiency of lidocaine base ethosomes remained stable for 60 days at 25°C ± 1°C (95% confidence interval [CI], -1.12% to 1.34%; P = 0.833). The transdermal flux of lidocaine base differed significantly for the 3 preparations (F = 120, P < 0.001), being significantly greater from ethosomes than from liposomes (95% corrected CI, 1129-1818 µg/(cm(2)·h); P < 0.001), and from hydroethanolic solution (95% corrected CI, 1468-2157 µg/(cm(2)·h); P < 0.001). Lidocaine base ethosomes had a shorter onset time and longer duration in vivo than did lidocaine base liposomes or lidocaine delivered in a hydroethanolic solution. Lidocaine base ethosomes showed no evidence of dermal irritation in guinea pigs. Ethosomes are potential carriers of local anesthetics across the skin and may have applicability for other percutaneous drugs that require rapid onset.

Chronic prenatal ethanol exposure increases adiposity and disrupts pancreatic morphology in adult guinea pig offspring.

PubMed

Dobson, C C; Mongillo, D L; Brien, D C; Stepita, R; Poklewska-Koziell, M; Winterborn, A; Holloway, A C; Brien, J F; Reynolds, J N

2012-12-17

Ethanol consumption during pregnancy can lead to a range of adverse developmental outcomes in children, termed fetal alcohol spectrum disorder (FASD). Central nervous system injury is a debilitating and widely studied manifestation of chronic prenatal ethanol exposure (CPEE). However, CPEE can also cause structural and functional deficits in metabolic pathways in offspring. This study tested the hypothesis that CPEE increases whole-body adiposity and disrupts pancreatic structure in guinea pig offspring. Pregnant guinea pigs received ethanol (4 g kg(-1) maternal body weight per day) or isocaloric-sucrose/pair-feeding (control) for 5 days per week throughout gestation. Male and female CPEE offspring demonstrated growth restriction at birth, followed by a rapid period of catch-up growth before weaning (postnatal day (PD) 1-7). Whole-body magnetic resonance imaging (MRI) in young adult offspring (PD100-140) revealed increased visceral and subcutaneous adiposity produced by CPEE. At the time of killing (PD150-200), CPEE offspring also had increased pancreatic adipocyte area and decreased β-cell insulin-like immunopositive area, suggesting reduced insulin production and/or secretion from pancreatic islets. CPEE causes increased adiposity and pancreatic dysmorphology in offspring, which may signify increased risk for the development of metabolic syndrome and type 2 diabetes mellitus.

[Historical review of insulin and its preparations in pharmacopoeia (3). Fish insulins].

PubMed

Suehiro, M

1992-01-01

Existence of encapsulated glands situated in the mesentery of certain teleosti was reported by Brockmann (1846) and Stannius (1848), respectively. Thus the gland was named stannius corpuscle or Brockmann body. Later, as results of histological study, cells of stannius corpuscle tissues were constituted with Langerhans islet cells observed in mammalian pancreas by Diammare (1899) and Laguesse (1906). Thus, before the days of discovery of insulin by Banting and Best in 1921, stannius corpuscle has been interesting from the aspects of comparative anatomy and physiology. Rennie (1906) examined a large number of specimens in various species of teleosti and gave the term "principal islet" to easily recognizable stannius corpuscle. Osawa studied comparative anatomy in Freiburg and returned to Tokyo. He continued the study of comparative anatomy of Langerhans islet aand published a report on observation of "principal islet" of flatfish, limanda yokohamae Gth. in 1912 in Japanese. His report seemed to be a milestone of studies of fish insulin in Japan. Macleod attempted to demonstrate direct evidence on secretion of insulin from Langerhans islet cells. Experiments were made on extraction of "principal islet" of teleosti, angler Lophius) and sculpin (Myoxocephalus) to obtain insulin and demonstrated activity. No insulin activity was obtained from pancreatic tissues constituted with acinar cells of these fishes. In the case of elasmobranch, Langerhans islets are not separated, but potent insulin could be extracted from the pancreas. His report published in 1922 was the first report on fish insulin. Succeeding to Macleod's report, several reports on fish insulin were contnributed from Canada, England and U.S.A. until 1929. Dr. Kkumagai, Professor of Internal Medicine, Tohoku Imperial University (Sendai) also conducted the studies on extraction of active principle of pancreas since 1920, independently. But, a Toronto group reached the goal on discovery of insulin earlier than the Sendai group. The Sendai group also described extraction of active principle from the "principal islet" of teleosti. Especially, Ukai (1926) described morphological study on pancreas and stannius corpuscle for more than twenty species of fish. His report played an important role as the next milestone on the road of fish insulin development studies in Japan. In 1926, Dr. Sakaguchi who was a leading clinical diabetologist in Japan published a monograph entitled "Insulin" written in Japanese. He referred the report on fish insulins of McCormick and Noble and Dr. Kumagai's report, however, he commented that production of insulin from fish seemed to be less worthy due to requirements of laborious work to collect small stannius corpuscle from fish. Professor A. Ogata described a textbook entitled "Zoki-Yakuhin-Kagaku (chemistry of organotherapeutics): in 1931. In the first edition, papers of Macleod, McCormick, Dudley and Osawa were referred. In the revised fifth edition (1940) contained description of unpublished data of insulin content of various kinds of fish caught in Japan and supplied from his student Nagasawa. Under the circumstance of expanding tendency of the China Incident to World War II, shortage of importation and production of insulin preparations manufactured from domestic animals was anticipated. Development on manufacture of fish insulin became urgent. [Truncated

Protection against lethal Marburg virus infection mediated by lipid encapsulated small interfering RNA.

PubMed

Ursic-Bedoya, Raul; Mire, Chad E; Robbins, Marjorie; Geisbert, Joan B; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W

2014-02-15

Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Treatment resulted in 60%-100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection.

Cross-validation of commercial enzyme-linked immunosorbent assay and radioimmunoassay for porcine C-peptide concentration measurements in non-human primate serum.

PubMed

Gresch, Sarah C; Mutch, Lucas A; Janecek, Jody L; Hegstad-Davies, Rebecca L; Graham, Melanie L

2017-09-01

C-peptide concentration is widely used as a marker of insulin secretion and is especially relevant in evaluating islet graft function following transplantation, because its measurement is not confounded by the presence of exogenous insulin. To address the shortage of human islet donors, the use of porcine islets has been proposed as a possible solution and the stringent pig-to-non-human primate (NHP) model is often the most relevant for pre-clinical evaluation of the potential for diabetes reversal resulting from an islet xenograft. The Millipore radioimmunoassay (RIA) was exclusively used to measure porcine C-peptide (PCP) until 2013 when the assay was discontinued and subsequently a commercially available enzyme-linked immunosorbent assay (ELISA) from Mercodia has been widely adopted. Both assays have been used in pre-clinical trials evaluating the therapeutic potential of xenograft products in reversing diabetes in the pig-to-NHP model, to interpret data in a comparable way it may be useful to perform a harmonization of C-peptide measurements. We performed a method comparison by determining the PCP concentration in 620 serum samples collected from 20 diabetic cynomolgus macaques transplanted with adult porcine islets. All analyses were performed according to manufacturer instructions. With both assays, we demonstrated an acceptable detection limit, precision, and recovery. Linearity of the ELISA met acceptance criteria at all concentrations tested while linearity of the RIA only met acceptance criteria at five of the eight concentrations tested. The RIA had a detection limit of 0.16 ng/mL, and recovery ranged from 82% to 96% and met linearity acceptance criteria at 0.35 ng/mL and from 0.78 to 2.33 ng/mL. The ELISA had a detection limit of 0.03 ng/mL, and recovery ranged from 81% to 115% and met linearity acceptance criteria from 0.08 to 0.85 ng/mL. Both assays had intra-assay precision <11% and inter-assay precision <14%. PCP concentration measured by ELISA demonstrated a significant correlation with RIA (R 2 =.9721, P<.0001). This strong correlation supports use of the regression equation y=2.029x+0.0897 to transform ELISA data to RIA or inversely y=0.4930x-0.0456 to convert RIA data to ELISA for direct comparison between assays in the concentration range of 0-3.0 ng/mL. Measured C-peptide concentration was lower with the ELISA than with the RIA; individual measurements plotted against the averages of the pair demonstrated that the variability from the mean strongly depended on increasing concentration. Porcine C-peptide can be reliably measured in NHP serum using the Mercodia ELISA, making this assay interchangeable with the Millipore RIA. Inherent differences in antibody affinity and calibration factors may explain the lower ELISA values as compared to the RIA; however without access to a traceable reference standard, it is not possible to determine which assay is most accurate. Regression modeling resulted in a correction factor appropriate for conversion of ELISA data to RIA-equivalent data facilitating comparison of assay results longitudinally and between groups. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Encapsulated Decon for Use on Medical Patients

DTIC Science & Technology

1983-12-01

Development of effective decon microcapsules was based on a series of tasks performed on this study. The preliminary tasks included a litera- ture search...culminated with evaluating selected microcapsules on pig skin samples, with HD, GB, arid GD. Results appear encouraging. The best capsule performance...term contact. in addition, a brief study showed magnetite can be incorporated into the capsule wall to provide magnetic microcapsules that can be

Successful immunosuppressant-free heterotopic transplantation of tracheal allografts in the pig.

PubMed

De Wolf, Julien; Brieu, Mathias; Zawadzki, Christophe; Ung, Alexandre; Kipnis, Eric; Jashari, Ramadan; Hubert, Thomas; Fayoux, Pierre; Mariette, Christophe; Copin, Marie-Christine; Wurtz, Alain

2017-08-01

It has been demonstrated that both heterotopic and orthotopic transplants of epithelium-denuded cryopreserved tracheal allografts are feasible in immunosuppressant-free rabbits. Validation of these results in large animals is required before considering clinical applications. We evaluated the viability, immune tolerance and strain properties of such tracheal allografts heterotopically transplanted in a pig model. Ten tracheal segments, 5 short (5 rings) and 5 long (10 rings), were obtained from male Landrace pigs. The tracheal segments were surgically denuded of their epithelium, then cryopreserved and stored in a tissue bank for 33 to 232 days. After thawing, tracheal segments stented with a silicone tube were wrapped in the omentum in 2 groups of 5 female recipients. The animals did not receive any immunosuppressive drugs. The animals were euthanized from Day 6 to Day 90 in both groups. An effective revascularization of allografts regardless of length was observed. Lymphocyte infiltrate was shown in the early postoperative period and became non-significant after 30 days. Allografts displayed high levels of neoangiogenesis and viable cartilage rings with islets of calcification. Biomechanical measurements demonstrated strain properties similar to those of a fresh tracheal segment from Day 58. Our results demonstrate the acceptability and satisfactory stiffness of epithelium-denuded cryopreserved tracheal allografts implanted in the omentum, despite the absence of immunosuppressive drugs. Since the omentum has the capability to reach the tracheal region, this approach should be investigated in the setting of orthotopic transplants in a pig model before considering clinical applications. © The Author 2017. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery. All rights reserved.

Marked augmentation of PLGA nanoparticle-induced metabolically beneficial impact of γ-oryzanol on fuel dyshomeostasis in genetically obese-diabetic ob/ob mice.

PubMed

Kozuka, Chisayo; Shimizu-Okabe, Chigusa; Takayama, Chitoshi; Nakano, Kaku; Morinaga, Hidetaka; Kinjo, Ayano; Fukuda, Kotaro; Kamei, Asuka; Yasuoka, Akihito; Kondo, Takashi; Abe, Keiko; Egashira, Kensuke; Masuzaki, Hiroaki

2017-11-01

Our previous works demonstrated that brown rice-specific bioactive substance, γ-oryzanol acts as a chaperone, attenuates exaggerated endoplasmic reticulum (ER) stress in brain hypothalamus and pancreatic islets, thereby ameliorating metabolic derangement in high fat diet (HFD)-induced obese diabetic mice. However, extremely low absorption efficiency from intestine of γ-oryzanol is a tough obstacle for the clinical application. Therefore, in this study, to overcome extremely low bioavailability of γ-oryzanol with super-high lipophilicity, we encapsulated γ-oryzanol in polymer poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Nano-Orz), and evaluated its metabolically beneficial impact in genetically obese-diabetic ob/ob mice, the best-known severest diabetic model in mice. To our surprise, Nano-Orz markedly ameliorated fuel metabolism with an unexpected magnitude (∼1000-fold lower dose) compared with regular γ-oryzanol. Furthermore, such a conspicuous impact was achievable by its administration once every 2 weeks. Besides the excellent impact on dysfunction of hypothalamus and pancreatic islets, Nano-Orz markedly decreased ER stress and inflammation in liver and adipose tissue. Collectively, nanotechnology-based developments of functional foods oriented toward γ-oryzanol shed light on the novel approach for the treatment of a variety of metabolic diseases in humans.

Use of the continuous glucose monitoring system in Goettingen Minipigs, with a special focus on the evaluation of insulin-dependent diabetes.

PubMed

Strauss, A; Tiurbe, C; Chodnevskaja, I; Thiede, A; Timm, S; Ulrichs, K; Moskalenko, V

2008-03-01

Adult pig islet isolation has greatly improved in the past few years. Islet grafts may now be tested in large animals. Continuous Glucose Monitoring System (CGMS) was applied to diabetic Goettingen Minipigs (GMP) to improve the management of hyperglycemia and hypoglycemia and their welfare before transplantation. GMP (25-35 kg) received a minipig diet once daily. Diabetes was induced by streptozotocin (STZ; 150 mg/kg intravenous [IV]; n = 5) or by surgical pancreatectomy (PGMP; n = 3). Interstitial glucose concentration (IGC) was monitored continuously with an implanted sensor; CGMS was calibrated using conventional blood glucose tests 3-4 times per day; CGMS data were fed into the monitor memory and analyzed using CGMS software. Glucose sensors were handled accurately. Diabetes occurred 2-3 days after STZ or immediately after pancreatectomy with basal C-peptide secretion of <0.4 ng/mL (measured using intravenous glucose tolerance test) and prompt loss of body weight. Insulin substitution was necessary to keep the GMP in good condition for up to 5-6 months, with stable body weight and normal behavior. Some GMP became hypoglycemic, which was only documented by CGMS, but not by conventional glucose assays. Tight glucose control and substitution of exocrine enzymes (Creon 25,000 E/d) reduced morbidity of the PGMP, which was then comparable with that of STZ-GMP. The CGMS, developed for humans, is equally suitable for the 2 GMP diabetes models. Close-meshed glucose monitoring and insulin treatment improved the general condition of the diabetic GMP, ie, the islet graft recipients, and will thus greatly add to posttransplantation success.

Protection Against Lethal Marburg Virus Infection Mediated by Lipid Encapsulated Small Interfering RNA

PubMed Central

Ursic-Bedoya, Raul; Mire, Chad E.; Robbins, Marjorie; Geisbert, Joan B.; Judge, Adam; MacLachlan, Ian; Geisbert, Thomas W.

2014-01-01

Background. Marburg virus (MARV) infection causes severe morbidity and mortality in humans and nonhuman primates. Currently, there are no licensed therapeutics available for treating MARV infection. Here, we present the in vitro development and in vivo evaluation of lipid-encapsulated small interfering RNA (siRNA) as a potential therapeutic for the treatment of MARV infection. Methods. The activity of anti-MARV siRNAs was assessed using dual luciferase reporter assays followed by in vitro testing against live virus. Lead candidates were tested in lethal guinea pig models of 3 different MARV strains (Angola, Ci67, Ravn). Results. Treatment resulted in 60%–100% survival of guinea pigs infected with MARV. Although treatment with siRNA targeting other MARV messenger RNA (mRNA) had a beneficial effect, targeting the MARV NP mRNA resulted in the highest survival rates. NP-718m siRNA in lipid nanoparticles provided 100% protection against MARV strains Angola and Ci67, and 60% against Ravn. A cocktail containing NP-718m and NP-143m provided 100% protection against MARV Ravn. Conclusions. These data show protective efficacy against the most pathogenic Angola strain of MARV. Further development of the lipid nanoparticle technology has the potential to yield effective treatments for MARV infection. PMID:23990568

Feasibility of Induced Pluripotent Stem Cell Therapies for Treatment of Type 1 Diabetes.

PubMed

Duffy, Caden; Prugue, Cesar; Glew, Rachel; Smith, Taryn; Howell, Calvin; Choi, Gina; Cook, Alonzo David

2018-06-27

Despite their potential for treating type 1 diabetes (T1D), induced pluripotent stem cells (iPSCs) have not yet been used successfully in the clinic. In this paper, advances in iPSC therapies are reviewed and compared to current methods of treating T1D. Encapsulation of iPSCs is being pursued to address such safety concerns as the possibility of immune rejection or teratoma formation, and provide for retrievability. Issues of material selection, cell differentiation, size of islet aggregates, sites of implantation, animal models, and vascularization are also being addressed. Clinical trials are being conducted to test a variety of new devices with the hope of providing additional therapies for T1D.

A single-islet microplate assay to measure mouse and human islet insulin secretion.

PubMed

Truchan, Nathan A; Brar, Harpreet K; Gallagher, Shannon J; Neuman, Joshua C; Kimple, Michelle E

2015-01-01

One complication to comparing β-cell function among islet preparations, whether from genetically identical or diverse animals or human organ donors, is the number of islets required per assay. Islet numbers can be limiting, meaning that fewer conditions can be tested; other islet measurements must be excluded; or islets must be pooled from multiple animals/donors for each experiment. Furthermore, pooling islets negates the possibility of performing single-islet comparisons. Our aim was to validate a 96-well plate-based single islet insulin secretion assay that would be as robust as previously published methods to quantify glucose-stimulated insulin secretion from mouse and human islets. First, we tested our new assay using mouse islets, showing robust stimulation of insulin secretion 24 or 48 h after islet isolation. Next, we utilized the assay to quantify mouse islet function on an individual islet basis, measurements that would not be possible with the standard pooled islet assay methods. Next, we validated our new assay using human islets obtained from the Integrated Islet Distribution Program (IIDP). Human islets are known to have widely varying insulin secretion capacity, and using our new assay we reveal biologically relevant factors that are significantly correlated with human islet function, whether displayed as maximal insulin secretion response or fold-stimulation of insulin secretion. Overall, our results suggest this new microplate assay will be a useful tool for many laboratories, expert or not in islet techniques, to be able to precisely quantify islet insulin secretion from their models of interest.

A Zebrafish Larval Model to Assess Virulence of Porcine Streptococcus suis Strains.

PubMed

Zaccaria, Edoardo; Cao, Rui; Wells, Jerry M; van Baarlen, Peter

2016-01-01

Streptococcus suis is an encapsulated Gram-positive bacterium, and the leading cause of sepsis and meningitis in young pigs resulting in considerable economic losses in the porcine industry. It is also considered an emerging zoonotic agent. In the environment, both avirulent and virulent strains occur in pigs, and virulent strains appear to cause disease in both humans and pigs. There is a need for a convenient, reliable and standardized animal model to assess S. suis virulence. A zebrafish (Danio rerio) larvae infection model has several advantages, including transparency of larvae, low cost, ease of use and exemption from ethical legislation up to 6 days post fertilization, but has not been previously established as a model for S. suis. Microinjection of different porcine strains of S. suis in zebrafish larvae resulted in highly reproducible dose- and strain-dependent larval death, strongly correlating with presence of the S. suis capsule and to the original virulence of the strain in pigs. Additionally we compared the virulence of the two-component system mutant of ciaRH, which is attenuated for virulence in both mice and pigs in vivo. Infection of larvae with the ΔciaRH strain resulted in significantly higher survival rate compared to infection with the S10 wild-type strain. Our data demonstrate that zebrafish larvae are a rapid and reliable model to assess the virulence of clinical porcine S. suis isolates.

A Zebrafish Larval Model to Assess Virulence of Porcine Streptococcus suis Strains

PubMed Central

Zaccaria, Edoardo; Cao, Rui; Wells, Jerry M.; van Baarlen, Peter

2016-01-01

Streptococcus suis is an encapsulated Gram-positive bacterium, and the leading cause of sepsis and meningitis in young pigs resulting in considerable economic losses in the porcine industry. It is also considered an emerging zoonotic agent. In the environment, both avirulent and virulent strains occur in pigs, and virulent strains appear to cause disease in both humans and pigs. There is a need for a convenient, reliable and standardized animal model to assess S. suis virulence. A zebrafish (Danio rerio) larvae infection model has several advantages, including transparency of larvae, low cost, ease of use and exemption from ethical legislation up to 6 days post fertilization, but has not been previously established as a model for S. suis. Microinjection of different porcine strains of S. suis in zebrafish larvae resulted in highly reproducible dose- and strain-dependent larval death, strongly correlating with presence of the S. suis capsule and to the original virulence of the strain in pigs. Additionally we compared the virulence of the two-component system mutant of ciaRH, which is attenuated for virulence in both mice and pigs in vivo. Infection of larvae with the ΔciaRH strain resulted in significantly higher survival rate compared to infection with the S10 wild-type strain. Our data demonstrate that zebrafish larvae are a rapid and reliable model to assess the virulence of clinical porcine S. suis isolates. PMID:26999052

Risk factors for islet loss during culture prior to transplantation.

PubMed

Kin, Tatsuya; Senior, Peter; O'Gorman, Doug; Richer, Brad; Salam, Abdul; Shapiro, Andrew Mark James

2008-11-01

Culturing islets can add great flexibility to a clinical islet transplant program. However, a reduction in the islet mass has been frequently observed during culture and its degree varies. The aim of this study was to identify the risk factors associated with a significant islet loss during culture. One-hundred and four islet preparations cultured in an attempt to use for transplantation constituted this study. After culture for 20 h (median), islet yield significantly decreased from 363 309 +/- 12 647 to 313 035 +/- 10 862 islet equivalent yield (IE) (mean +/- SE), accompanied by a reduction in packed tissue volume from 3.9 +/- 0.1 to 3.0 +/- 0.1 ml and islet index (IE/islet particle count) from 1.20 +/- 0.04 to 1.05 +/- 0.04. Culture did not markedly alter islet purity or percent of trapped islet. Morphology score and viability were significantly improved after culture. Of 104 islet preparations, 37 suffered a substantial islet loss (> 20%) over culture. Factors significantly associated with risk of islet loss identified by univariate analysis were longer cold ischemia time, two-layer method (TLM) preservation, lower islet purity, and higher islet index. Multivariate analysis revealed that independent predictors of islet loss were higher islet index and the use of TLM. This study provides novel information on the link between donor- isolation factors and islet loss during culture.

Glutathione Ethyl Ester Supplementation during Pancreatic Islet Isolation Improves Viability and Transplant Outcomes in a Murine Marginal Islet Mass Model

PubMed Central

Raposo do Amaral, Alexandre S.; Pawlick, Rena L.; Rodrigues, Erika; Costal, Flavia; Pepper, Andrew; Ferreira Galvão, Flávio H.; Correa-Giannella, Maria Lucia; Shapiro, A. M.James

2013-01-01

Background The success of pancreatic islet transplantation still faces many challenges, mainly related to cell damage during islet isolation and early post-transplant. The increased generation of reactive oxygen species (ROS) during islet isolation and the consumption of antioxidant defenses appear to be an important pathway related to islet damage. Methodology/Principal Findings In the present study we evaluated whether supplementation of glutathione-ethyl-ester (GEE) during islet isolation could improve islet viability and transplant outcomes in a murine marginal islet mass model. We also cultured human islets for 24 hours in standard CMRL media with or without GEE supplementation. Supplementation of GEE decreased the content of ROS in isolated islets, leading to a decrease in apoptosis and maintenance of islet viability. A higher percentage of mice transplanted with a marginal mass of GEE treated islets became euglycemic after transplant. The supplementation of 20 mM GEE in cultured human islets significantly reduced the apoptosis rate in comparison to untreated islets. Conclusions/Significance GEE supplementation was able to decrease the apoptosis rate and intracellular content of ROS in isolated islets and might be considered a potential intervention to improve islet viability during the isolation process and maintenance in culture before islet transplantation. PMID:23424628

Effect of immunodepletion of MHC class II-positive cells from pancreatic islets on generation of cytotoxic T-lymphocytes in mixed islet-lymphocyte coculture.

PubMed

Stock, P G; Ascher, N L; Platt, J L; Kaufman, D B; Chen, S; Field, M J; Sutherland, D E

1989-01-01

In vitro manipulation of pancreatic islets to decrease islet immunogenicity before transplantation has largely been directed at eliminating the major histocompatibility complex (MHC) class II-positive passenger leukocytes from the islets. The mixed islet-lymphocyte coculture (MILC) system was used to quantitate the efficacy of immunodepletion of MHC class II-positive cells from pancreatic islets in terms of reducing immunogenicity. With these experiments we compared the in vitro immunogenicity of MHC class II-depleted islets with untreated islets. B10.BR (H-2k) islets were treated with anti-Iak alloserum followed by complement. This treatment successfully eliminated MHC class II-positive cells from the islets, as demonstrated by indirect immunofluorescence techniques. Depleted islets generated slightly lower amounts of allospecific cytotoxic T-lymphocyte (CTL) activity when exposed to C57BL/6 (H-2b) splenocytes in the MILC than untreated control islets. Although the amount of CTL generated by the depleted islets was slightly less than that generated by untreated islets, there was significant stimulation of CTL by the MHC class II-depleted islets. Therefore, the presence or absence of MHC class II cells within the islet is unlikely to be the decisive factor contributing to islet immunogenicity.

Determination of Optimal Sample Size for Quantification of β-Cell Area, Amyloid Area and β-Cell Apoptosis in Isolated Islets.

PubMed

Meier, Daniel T; Entrup, Leon; Templin, Andrew T; Hogan, Meghan F; Samarasekera, Thanya; Zraika, Sakeneh; Boyko, Edward J; Kahn, Steven E

2015-08-01

Culture of isolated rodent islets is widely used in diabetes research to assess different endpoints, including outcomes requiring histochemical staining. As islet yields during isolation are limited, we determined the number of islets required to obtain reliable data by histology. We found that mean values for insulin-positive β-cell area/islet area, thioflavin S-positive amyloid area/islet area and β-cell apoptosis do not vary markedly when more than 30 islets are examined. Measurement variability declines as more islets are quantified, so that the variability of the coefficient of variation (CV) in human islet amyloid polypeptide (hIAPP) transgenic islets for β-cell area/islet area, amyloid area/islet area and β-cell apoptosis are 13.20% ± 1.52%, 10.03% ± 1.76% and 6.78% ± 1.53%, respectively (non-transgenic: 7.65% ± 1.17% β-cell area/islet area and 8.93% ± 1.56% β-cell apoptosis). Increasing the number of islets beyond 30 had marginal effects on the CV. Using 30 islets, 6 hIAPP-transgenic preparations are required to detect treatment effects of 14% for β-cell area/islet area, 30% for amyloid area/islet area and 23% for β-cell apoptosis (non-transgenic: 9% for β-cell area/islet area and 45% for β-cell apoptosis). This information will be of value in the design of studies using isolated islets to examine β cells and islet amyloid. © The Author(s) 2015.

Oxygen environment and islet size are the primary limiting factors of isolated pancreatic islet survival

PubMed Central

Komatsu, Hirotake; Cook, Colin; Wang, Chia-Hao; Medrano, Leonard; Lin, Henry; Kandeel, Fouad; Tai, Yu-Chong; Mullen, Yoko

2017-01-01

Background Type 1 diabetes is an autoimmune disease that destroys insulin-producing beta cells in the pancreas. Pancreatic islet transplantation could be an effective treatment option for type 1 diabetes once several issues are resolved, including donor shortage, prevention of islet necrosis and loss in pre- and post-transplantation, and optimization of immunosuppression. This study seeks to determine the cause of necrotic loss of isolated islets to improve transplant efficiency. Methodology The oxygen tension inside isolated human islets of different sizes was simulated under varying oxygen environments using a computational in silico model. In vitro human islet viability was also assessed after culturing in different oxygen conditions. Correlation between simulation data and experimentally measured islet viability was examined. Using these in vitro viability data of human islets, the effect of islet diameter and oxygen tension of the culture environment on islet viability was also analyzed using a logistic regression model. Principal findings Computational simulation clearly revealed the oxygen gradient inside the islet structure. We found that oxygen tension in the islet core was greatly lower (hypoxic) than that on the islet surface due to the oxygen consumption by the cells. The hypoxic core was expanded in the larger islets or in lower oxygen cultures. These findings were consistent with results from in vitro islet viability assays that measured central necrosis in the islet core, indicating that hypoxia is one of the major causes of central necrosis. The logistic regression analysis revealed a negative effect of large islet and low oxygen culture on islet survival. Conclusions/Significance Hypoxic core conditions, induced by the oxygen gradient inside islets, contribute to the development of central necrosis of human isolated islets. Supplying sufficient oxygen during culture could be an effective and reasonable method to maintain isolated islets viable. PMID:28832685

Liposomal lidocaine gel for topical use at the oral mucosa: characterization, in vitro assays and in vivo anesthetic efficacy in humans.

PubMed

Franz-Montan, Michelle; Baroni, Daniela; Brunetto, Giovana; Sobral, Viviane Roberta Vieira; da Silva, Camila Morais Gonçalves; Venâncio, Paulo; Zago, Patricia Wiziack; Cereda, Cintia Maria Saia; Volpato, Maria Cristina; de Araújo, Daniele Ribeiro; de Paula, Eneida; Groppo, Francisco Carlos

2015-03-01

To characterize liposomal-lidocaine formulations for topical use on oral mucosa and to compare their in vitro permeation and in vivo anesthetic efficacy with commercially available lidocaine formulations. Large unilamellar liposomes (400 nm) containing lidocaine were prepared using phosphatidylcholine, cholesterol, and α-tocoferol (4:3:0.07, w:w:w) and were characterized in terms of membrane/water partition coefficient, encapsulation efficiency, size, polydispersity, zeta potential, and in vitro release. In vitro permeation across pig palatal mucosa and in vivo topical anesthetic efficacy on the palatal mucosa in healthy volunteers (double-blinded cross-over, placebo controlled study) were performed. The following formulations were tested: liposome-encapsulated 5% lidocaine (Liposome-Lido5); liposome-encapsulated 2.5% lidocaine (Liposome-Lido2.5); 5% lidocaine ointment (Xylocaina®), and eutectic mixture of lidocaine and prilocaine 2.5% (EMLA®). The Liposome-Lido5 and EMLA showed the best in vitro permeation parameters (flux and permeability coefficient) in comparison with Xylocaina and placebo groups, as well as the best in vivo topical anesthetic efficacy. We successfully developed and characterized a liposome encapsulated 5% lidocaine gel. It could be considered an option to other topical anesthetic agents for oral mucosa.

Seaweed extracts and galacto-oligosaccharides improve intestinal health in pigs following Salmonella Typhimurium challenge.

PubMed

Bouwhuis, M A; McDonnell, M J; Sweeney, T; Mukhopadhya, A; O'Shea, C J; O'Doherty, J V

2017-09-01

Pork and pork products are recognised as vehicles of Salmonella Typhimurium infection in humans. Seaweed-derived polysaccharides (SWE) and galacto-oligosaccharides (GOS) have shown to exhibit antimicrobial, prebiotic and immunomodulatory activity. The objective of this study was to assess the effects of dietary GOS and SWE supplementation on reducing S. Typhimurium numbers and intestinal inflammation in vivo. In total, 30 pigs (n=10/treatment, BW 30.9 kg) were randomly assigned to three dietary treatments: (1) basal diet; (2) basal diet+2.5 g GOS/kg diet; (3) basal diet+SWE (containing 180 mg laminarin/kg diet+340 mg fucoidan/kg diet). Following an 11-day dietary adaptation period, pigs were orally challenged with 108 colony-forming units/ml S. Typhimurium (day 0). Pigs remained on their diets for a further 17 days and were then sacrificed for sample collection. The SWE supplementation did not affect S. Typhimurium numbers on days 2 and 4 post-challenge but reduced S. Typhimurium numbers in faecal samples collected day 7 post-challenge (-0.80 log gene copy numbers (GCN)/g faeces) and in caecal and colonic digesta (-0.62 and -0.98 log GCN/g digesta, respectively; P<0.05) compared with the control treatment. Lactobacillus numbers were increased in caecal and colonic digesta after GOS supplementation (+0.70 and +0.35 log GCN/g digesta, respectively; P<0.05). In colonic tissue, both GOS and SWE supplementation resulted in reduced messenger RNA expression levels of interleukin (IL)-6, IL-22, tumour necrosis factor-α and regenerating islet-derived protein 3-γ (P<0.05). It can be concluded that dietary supplementation of SWE reduced faecal and intestinal S. Typhimurium numbers compared with the basal diet, whereas dietary GOS supplementation increased Lactobacillus numbers in caecal and colonic digesta but did not affect S. Typhimurium numbers. Supplementation of GOS and SWE reduced the gene expression of pro-inflammatory cytokines in colonic tissue of pigs after the experimental S. Typhimurium challenge.

Automated separation of merged Langerhans islets

NASA Astrophysics Data System (ADS)

Švihlík, Jan; Kybic, Jan; Habart, David

2016-03-01

This paper deals with separation of merged Langerhans islets in segmentations in order to evaluate correct histogram of islet diameters. A distribution of islet diameters is useful for determining the feasibility of islet transplantation in diabetes. First, the merged islets at training segmentations are manually separated by medical experts. Based on the single islets, the merged islets are identified and the SVM classifier is trained on both classes (merged/single islets). The testing segmentations were over-segmented using watershed transform and the most probable back merging of islets were found using trained SVM classifier. Finally, the optimized segmentation is compared with ground truth segmentation (correctly separated islets).

Biodegradable nanoparticle delivery of inactivated swine influenza virus vaccine provides heterologous cell-mediated immune response in pigs.

PubMed

Dhakal, Santosh; Hiremath, Jagadish; Bondra, Kathryn; Lakshmanappa, Yashavanth S; Shyu, Duan-Liang; Ouyang, Kang; Kang, Kyung-Il; Binjawadagi, Basavaraj; Goodman, Jonathan; Tabynov, Kairat; Krakowka, Steven; Narasimhan, Balaji; Lee, Chang Won; Renukaradhya, Gourapura J

2017-02-10

Swine influenza virus (SwIV) is one of the important zoonotic pathogens. Current flu vaccines have failed to provide cross-protection against evolving viruses in the field. Poly(lactic-co-glycolic acid) (PLGA) is a biodegradable FDA approved polymer and widely used in drug and vaccine delivery. In this study, inactivated SwIV H1N2 antigens (KAg) encapsulated in PLGA nanoparticles (PLGA-KAg) were prepared, which were spherical in shape with 200 to 300nm diameter, and induced maturation of antigen presenting cells in vitro. Pigs vaccinated twice with PLGA-KAg via intranasal route showed increased antigen specific lymphocyte proliferation and enhanced the frequency of T-helper/memory and cytotoxic T cells (CTLs) in peripheral blood mononuclear cells (PBMCs). In PLGA-KAg vaccinated and heterologous SwIV H1N1 challenged pigs, clinical flu symptoms were absent, while the control pigs had fever for four days. Grossly and microscopically, reduced lung pathology and viral antigenic mass in the lung sections with clearance of infectious challenge virus in most of the PLGA-KAg vaccinated pig lung airways were observed. Immunologically, PLGA-KAg vaccine irrespective of not significantly boosting the mucosal antibody response, it augmented the frequency of IFN-γ secreting total T cells, T-helper and CTLs against both H1N2 and H1N1 SwIV. In summary, inactivated influenza virus delivered through PLGA-NPs reduced the clinical disease and induced cross-protective cell-mediated immune response in a pig model. Our data confirmed the utility of a pig model for intranasal particulate flu vaccine delivery platform to control flu in humans. Copyright © 2017 Elsevier B.V. All rights reserved.

Factor Affecting Transplant Outcomes in Diabetic Nude Mice Receiving Human, Porcine, and Non-Human Primate Islets: Analysis of 335 Transplantations

PubMed Central

Loganathan, Gopalakrishnan; Graham, Melanie L.; Radosevich, David M.; Soltani, Sajjad M.; Tiwari, Mukesh; Anazawa, Takayuki; papas, Klearchos K.; Sutherland, David E.R.; Hering, Bernhard J.; Balamurugan, A.N.

2013-01-01

Background In the absence of a reliable islet potency assay, nude mice transplant is the criterion standard to assess islet quality for clinical transplantation. There are factors other than islet quality that affect the transplant outcome. Methods Here, we analyzed the transplant outcomes in 335 nude mice (NM) receiving islets from human (n=103), porcine (n=205), and non-human primate (NHP) donors (n=27). The islets (750, 1000, and 2000 islet equivalents) were transplanted under the kidney capsule of streptozotocin (STZ) induced diabetic NM. Results The proportion of mice that achieved normoglycemia was significantly higher in the group implanted with 2000 IEQ of human, porcine, or NHP islets (75% normoglycemic) versus groups that were implanted with 750 IEQ (7% normoglycemic) and 1000 IEQ (30% normoglycemic). In this study, we observed that the purity of porcine islet preparations (P ≤ .001), islet pellet size in porcine preparations (P ≤ .01) and mice recipient body weight for human islets preparations (P =.013), was independently associated with successful transplant outcome. NHP islets of 1000 IEQ were sufficient to achieve normoglycemic condition (83%). An islet mass of 2000 IEQ, high islet purity, increased recipient body weight, and high islet pellet volume increased the likelihood of successful reversal of diabetes in transplanted mice. Also, higher insulin secretory status of islets at basal stimulus was associated with a reduced mouse cure rate. The cumulative incidence of graft failure was significantly greater in human islets (56.12%) compared with porcine islets 35.57% (P ≤ .001). Conclusion Factors affecting NM bioassay were identified (islet mass, islet purity, pellet size, in vitro insulin secretory capability and mouse recipient body weight) and should be considered when evaluating islet function. PMID:23677052

Benefits of PEGylation in the early post-transplant period of intraportal islet transplantation as assessed by magnetic resonance imaging of labeled islets.

PubMed

Jin, Sang-Man; Oh, Seung-Hoon; Oh, Bae Jun; Suh, Sunghwan; Bae, Ji Cheol; Lee, Jung Hee; Lee, Myung-Shik; Lee, Moon-Kyu; Kim, Kwang-Won; Kim, Jae Hyeon

2014-01-01

While a few studies have demonstrated the benefit of PEGylation in islet transplantation, most have employed renal subcapsular models and none have performed direct comparisons of islet mass in intraportal islet transplantation using islet magnetic resonance imaging (MRI). In this study, our aim was to demonstrate the benefit of PEGylation in the early post-transplant period of intraportal islet transplantation with a novel algorithm for islet MRI. Islets were PEGylated after ferucarbotran labeling in a rat syngeneic intraportal islet transplantation model followed by comparisons of post-transplant glycemic levels in recipient rats infused with PEGylated (n = 12) and non-PEGylated (n = 13) islets. The total area of hypointense spots and the number of hypointense spots larger than 1.758 mm(2) of PEGylated and non-PEGylated islets were quantitatively compared. The total area of hypointense spots (P < 0.05) and the number of hypointense spots larger than 1.758 mm(2) (P < 0.05) were higher in the PEGylated islet group 7 and 14 days post translation (DPT). These results translated into better post-transplant outcomes in the PEGylated islet group 28 DPT. In validation experiments, MRI parameters obtained 1, 7, and 14 DPT predicted normoglycemia 4 wk post-transplantation. We directly demonstrated the benefit of islet PEGylation in protection against nonspecific islet destruction in the early post-transplant period of intraportal islet transplantation using a novel algorithm for islet MRI. This novel algorithm could serve as a useful tool to demonstrate such benefit in future clinical trials of islet transplantation using PEGylated islets.

Noninvasive imaging of islet grafts using positron-emission tomography

NASA Astrophysics Data System (ADS)

Lu, Yuxin; Dang, Hoa; Middleton, Blake; Zhang, Zesong; Washburn, Lorraine; Stout, David B.; Campbell-Thompson, Martha; Atkinson, Mark A.; Phelps, Michael; Gambhir, Sanjiv Sam; Tian, Jide; Kaufman, Daniel L.

2006-07-01

Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. However, islet transplantation is not routinely successful because most islet recipients gradually lose graft function. Furthermore, serological markers of islet function are insensitive to islet loss until the latter stages of islet graft rejection. A noninvasive method of monitoring islet grafts would aid in the assessment of islet graft survival and the evaluation of interventions designed to prolong graft survival. Here, we show that recombinant adenovirus can engineer isolated islets to express a positron-emission tomography (PET) reporter gene and that these islets can be repeatedly imaged by using microPET after transplantation into mice. The magnitude of signal from engineered islets implanted into the axillary cavity was directly related to the implanted islet mass. PET signals attenuated over the following weeks because of the transient nature of adenovirus-mediated gene expression. Because the liver is the preferred site for islet implantation in humans, we also tested whether islets could be imaged after transfusion into the mouse liver. Control studies revealed that both intrahepatic islet transplantation and hyperglycemia altered the biodistribution kinetics of the PET probe systemically. Although transplanted islets were dispersed throughout the liver, clear signals from the liver region of mice receiving PET reporter-expressing islets were detectable for several weeks. Viral transduction, PET reporter expression, and repeated microPET imaging had no apparent deleterious effects on islet function after implantation. These studies lay a foundation for noninvasive quantitative assessments of islet graft survival using PET. diabetes | transplantation

Intraportal islet oxygenation.

PubMed

Suszynski, Thomas M; Avgoustiniatos, Efstathios S; Papas, Klearchos K

2014-05-01

Islet transplantation (IT) is a promising therapy for the treatment of diabetes. The large number of islets required to achieve insulin independence limit its cost-effectiveness and the number of patients who can be treated. It is believed that >50% of islets are lost in the immediate post-IT period. Poor oxygenation in the early post-IT period is recognized as a possible reason for islet loss and dysfunction but has not been extensively studied. Several key variables affect oxygenation in this setting, including (1) local oxygen partial pressure (pO(2)), (2) islet oxygen consumption, (3) islet size (diameter, D), and (4) presence or absence of thrombosis on the islet surface. We discuss implications of oxygen-limiting conditions on intraportal islet viability and function. Of the 4 key variables, the islet size appears to be the most important determinant of the anoxic and nonfunctional islet volume fractions. Similarly, the effect of thrombus formation on the islet surface may be substantial. At the University of Minnesota, average size distribution data from clinical alloislet preparations (n = 10) indicate that >150-µm D islets account for only ~30% of the total islet number, but >85% of the total islet volume. This suggests that improved oxygen supply to the islets may have a profound impact on islet survivability and function since most of the β-cell volume is within large islets which are most susceptible to oxygen-limiting conditions. The assumption that the liver is a suitable islet transplant site from the standpoint of oxygenation should be reconsidered. © 2014 Diabetes Technology Society.

Intraportal Islet Oxygenation

PubMed Central

Suszynski, Thomas M.; Avgoustiniatos, Efstathios S.

2014-01-01

Islet transplantation (IT) is a promising therapy for the treatment of diabetes. The large number of islets required to achieve insulin independence limit its cost-effectiveness and the number of patients who can be treated. It is believed that >50% of islets are lost in the immediate post-IT period. Poor oxygenation in the early post-IT period is recognized as a possible reason for islet loss and dysfunction but has not been extensively studied. Several key variables affect oxygenation in this setting, including (1) local oxygen partial pressure (pO2), (2) islet oxygen consumption, (3) islet size (diameter, D), and (4) presence or absence of thrombosis on the islet surface. We discuss implications of oxygen-limiting conditions on intraportal islet viability and function. Of the 4 key variables, the islet size appears to be the most important determinant of the anoxic and nonfunctional islet volume fractions. Similarly, the effect of thrombus formation on the islet surface may be substantial. At the University of Minnesota, average size distribution data from clinical alloislet preparations (n = 10) indicate that >150-µm D islets account for only ~30% of the total islet number, but >85% of the total islet volume. This suggests that improved oxygen supply to the islets may have a profound impact on islet survivability and function since most of the β-cell volume is within large islets which are most susceptible to oxygen-limiting conditions. The assumption that the liver is a suitable islet transplant site from the standpoint of oxygenation should be reconsidered. PMID:24876622

Islet product characteristics and factors related to successful human islet transplantation from the Collaborative Islet Transplant Registry (CITR) 1999-2010.

PubMed

Balamurugan, A N; Naziruddin, B; Lockridge, A; Tiwari, M; Loganathan, G; Takita, M; Matsumoto, S; Papas, K; Trieger, M; Rainis, H; Kin, T; Kay, T W; Wease, S; Messinger, S; Ricordi, C; Alejandro, R; Markmann, J; Kerr-Conti, J; Rickels, M R; Liu, C; Zhang, X; Witkowski, P; Posselt, A; Maffi, P; Secchi, A; Berney, T; O'Connell, P J; Hering, B J; Barton, F B

2014-11-01

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999-2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p<0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007-2010 when compared to 1999-2002 (445.4±156.8 vs. 421.3±155.4×0(3) IEQ; p<0.05). Islet purity and total number of β cells significantly improved over the study period (p<0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999-2010, and these parallel improvements in clinical outcomes over the same period. © 2014 The Authors. American Journal of Transplantation Published by Wiley Periodicals, Inc. on behalf of American Society of Transplant Surgeons.

Islet Product Characteristics and Factors Related to Successful Human Islet Transplantation From the Collaborative Islet Transplant Registry (CITR) 1999–2010

PubMed Central

Balamurugan, A N; Naziruddin, B; Lockridge, A; Tiwari, M; Loganathan, G; Takita, M; Matsumoto, S; Papas, K; Trieger, M; Rainis, H; Kin, T; Kay, T W; Wease, S; Messinger, S; Ricordi, C; Alejandro, R; Markmann, J; Kerr-Conti, J; Rickels, M R; Liu, C; Zhang, X; Witkowski, P; Posselt, A; Maffi, P; Secchi, A; Berney, T; O’Connell, P J; Hering, B J; Barton, F B

2014-01-01

The Collaborative Islet Transplant Registry (CITR) collects data on clinical islet isolations and transplants. This retrospective report analyzed 1017 islet isolation procedures performed for 537 recipients of allogeneic clinical islet transplantation in 1999–2010. This study describes changes in donor and islet isolation variables by era and factors associated with quantity and quality of final islet products. Donor body weight and BMI increased significantly over the period (p < 0.001). Islet yield measures have improved with time including islet equivalent (IEQ)/particle ratio and IEQs infused. The average dose of islets infused significantly increased in the era of 2007–2010 when compared to 1999–2002 (445.4 ± 156.8 vs. 421.3 ± 155.4 ×103 IEQ; p < 0.05). Islet purity and total number of β cells significantly improved over the study period (p < 0.01 and <0.05, respectively). Otherwise, the quality of clinical islets has remained consistently very high through this period, and differs substantially from nonclinical islets. In multivariate analysis of all recipient, donor and islet factors, and medical management factors, the only islet product characteristic that correlated with clinical outcomes was total IEQs infused. This analysis shows improvements in both quantity and some quality criteria of clinical islets produced over 1999–2010, and these parallel improvements in clinical outcomes over the same period. PMID:25278159

Inflammatory Response in Islet Transplantation

PubMed Central

Kanak, Mazhar A.; Kunnathodi, Faisal; Lawrence, Michael C.; Levy, Marlon F.

2014-01-01

Islet cell transplantation is a promising beta cell replacement therapy for patients with brittle type 1 diabetes as well as refractory chronic pancreatitis. Despite the vast advancements made in this field, challenges still remain in achieving high frequency and long-term successful transplant outcomes. Here we review recent advances in understanding the role of inflammation in islet transplantation and development of strategies to prevent damage to islets from inflammation. The inflammatory response associated with islets has been recognized as the primary cause of early damage to islets and graft loss after transplantation. Details on cell signaling pathways in islets triggered by cytokines and harmful inflammatory events during pancreas procurement, pancreas preservation, islet isolation, and islet infusion are presented. Robust control of pre- and peritransplant islet inflammation could improve posttransplant islet survival and in turn enhance the benefits of islet cell transplantation for patients who are insulin dependent. We discuss several potent anti-inflammatory strategies that show promise for improving islet engraftment. Further understanding of molecular mechanisms involved in the inflammatory response will provide the basis for developing potent therapeutic strategies for enhancing the quality and success of islet transplantation. PMID:24883060

The effect of curcumin on insulin release in rat-isolated pancreatic islets.

PubMed

Abdel Aziz, Mohamed T; El-Asmar, Mohamed F; El Nadi, Essam G; Wassef, Mohamed A; Ahmed, Hanan H; Rashed, Laila A; Obaia, Eman M; Sabry, Dina; Hassouna, Amira A; Abdel Aziz, Ahmed T

2010-08-01

Curcumin exerts a hypoglycemic action and induces heme-oxygenase-1 (HO-1). We evaluated the effect of curcumin on isolated islets of Langerhans and studied whether its action on insulin secretion is mediated by inducible HO-1. Islets were isolated from rats and divided into control islets, islets incubated in different curcumin concentrations, islets incubated in hemin, islets incubated in curcumin and HO inhibitor, stannous mesoporphyrin (SnMP), islets incubated in hemin and SnMP, islets incubated in SnMP only, and islets incubated in 16.7 mmol/L glucose. Heme-oxygenase activity, HO-1 expression, and insulin estimation was assessed. Insulin secretion, HO-1 gene expression and HO activity were significantly increased in islets incubated in curcumin, hemin, and glucose compared with controls. This increase in insulin secretion was significantly decreased by incubation of islets in SnMP. The action of curcumin on insulin secretion from the isolated islets may be, in part, mediated through increased HO-1 gene expression.

Repair of pig dura in vivo using temperature controlled CO(2) laser soldering.

PubMed

Forer, Boaz; Vasilyev, Tamar; Brosh, Tamar; Kariv, Noam; Gil, Ziv; Fliss, Dan M; Katzir, Abraham

2005-10-01

The purpose of this study was to demonstrate that laser soldering might be successfully used for closing holes or cuts in the dura layer, which encapsulates the brain. A temperature controlled fiberoptic CO(2) laser system and albumin solder were used for spot soldering of fascia patches to holes in the dura of farm pigs, in vitro and in vivo. The mean burst pressure of the soldered patches in the in vitro experiments was 190 +/- 88 mm Hg-significantly higher than typical maximum CSF pressure of 15 mm Hg. In the in vivo experiments the pigs showed no postoperative complications. Histopathological studies exhibited an accepted level of inflammatory reaction and showed no thermal damage to the underlying brain tissue. It has been clearly demonstrated that temperature controlled laser soldering is a very useful technique for the repair of the dura. It provides significant advantages over standard closure techniques: it is easy to apply, the bond is strong and watertight and the procedure is likely to be much faster than suturing. This research work will lead to clinical trials.

A BRIEF HISTORY OF CLINICAL XENOTRANSPLANTATION

PubMed Central

Cooper, David K. C.; Ekser, Burcin; Tector, A. Joseph

2015-01-01

Between the 17th and 20th centuries, blood was transfused from various animal species into patients with a variety of pathological conditions. Skin grafts were carried out in the 19th century, with grafts from a variety of animals, with frogs being the most popular. In the 1920s, Voronoff advocated the transplantation of slices of chimpanzee testis into elderly men, believing that the hormones produced by the testis would rejuvenate his patients. In 1963–4, when human organs were not available and dialysis was not yet in use, Reemtsma transplanted chimpanzee kidneys into 13 patients, one of whom returned to work for almost 9 months before suddenly dying from what was believed to be an electrolyte disturbance. The first heart transplant in a human ever performed was by Hardy in 1964, using a chimpanzee heart, but the patient died within two hours. Starzl carried out the first chimpanzee-to-human liver transplantation in 1966; in 1992 he obtained patient survival for 70 days following a baboon liver transplant. The first clinical pig islet transplant was carried out by Groth in 1993. Today, genetically-modified pigs offer hope of a limitless supply of organs and cells for those in need of a transplant. PMID:26118617

Optimal formation of genetically modified and functional pancreatic islet spheroids by using hanging-drop strategy.

PubMed

Kim, H J; Alam, Z; Hwang, J W; Hwang, Y H; Kim, M J; Yoon, S; Byun, Y; Lee, D Y

2013-03-01

Rejection and hypoxia are important factors causing islet loss at an early stage after pancreatic islet transplantation. Recently, islets have been dissociated into single cells for reaggregation into so-called islet spheroids. Herein, we used a hanging-drop strategy to form islet spheroids to achieve functional equivalence to intact islets. To obtain single islet cells, we dissociated islets with trypsin-EDTA digestion for 10 minutes. To obtain spheroids, we dropped various numbers of single cells (125, 250, or 500 cells/30 μL drop) onto a Petri dish, that was inverted for incubation in humidified air containing 5% CO(2) at 37 °C for 7 days. The aggregated spheroids in the droplets were harvested for further culture. The size of the aggregated islet spheroids depended on the number of single cells (125-500 cells/30 μL droplet). Their morphology was similar to that of intact islets without any cellular damage. When treated with various concentrations of glucose to evaluate responsiveness, their glucose-mediated stimulation index value was similar to that of intact islets, an observation that was attributed to strong cell-to-cell interactions in islet spheroids. However, islet spheroids aggregated in general culture dishes showed abnormal glucose responsiveness owing to weak cell-to-cell interactions. Cell-to-cell interactions in islet spheroids were confirmed with an anti-connexin-36 monoclonal antibody. Finally, nonviral poly(ethylene imine)-mediated interleukin-10 cytokine gene delivered beforehand into dissociated single cells before formation of islet spheroids increased the gene transfection efficacy and interleukin-10 secretion from islet spheroids >4-fold compared with intact islets. These results demonstrated the potential application of genetically modified, functional islet spheroids with of controlled size and morphology using an hanging-drop technique. Copyright © 2013 Elsevier Inc. All rights reserved.

Reduction of diffusion barriers in isolated rat islets improves survival, but not insulin secretion or transplantation outcome

PubMed Central

Janette Williams, S; Huang, Han-Hung; Kover, Karen; Moore, Wayne; Berkland, Cory; Singh, Milind; Smirnova, Irina V; MacGregor, Ronal

2010-01-01

For people with type 1 diabetes and severe hypoglycemic unawareness, islet transplants offer hope for improving the quality of life. However, islet cell death occurs quickly during or after transplantation, requiring large quantities of islets per transplant. The purpose of this study was to determine whether poor function demonstrated in large islets was a result of diffusion barriers and if removing those barriers could improve function and transplantation outcomes. Islets were isolated from male DA rats and measured for cell viability, islet survival, glucose diffusion and insulin secretion. Modeling of diffusion barriers was completed using dynamic partial differential equations for a sphere. Core cell death occurred in 100% of the large islets (diameter >150 µm), resulting in poor survival within 7 days after isolation. In contrast, small islets (diameter <100 µm) exhibited good survival rates in culture (91%). Glucose diffusion into islets was tracked with 2-NBDG; 4.2 µm/min in small islets and 2.8 µm/min in large islets. 2-NBDG never permeated to the core cells of islets larger than 150 µm diameter. Reducing the diffusion barrier in large islets improved their immediate and long-term viability in culture. However, reduction of the diffusion barrier in large islets failed to improve their inferior in vitro insulin secretion compared to small islets, and did not return glucose control to diabetic animals following transplantation. Thus, diffusion barriers lead to low viability and poor survival for large islets, but are not solely responsible for the inferior insulin secretion or poor transplantation outcomes of large versus small islets. PMID:20885858

Automated digital image analysis of islet cell mass using Nikon's inverted eclipse Ti microscope and software to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

PubMed

Gmyr, Valery; Bonner, Caroline; Lukowiak, Bruno; Pawlowski, Valerie; Dellaleau, Nathalie; Belaich, Sandrine; Aluka, Isanga; Moermann, Ericka; Thevenet, Julien; Ezzouaoui, Rimed; Queniat, Gurvan; Pattou, Francois; Kerr-Conte, Julie

2015-01-01

Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method (p < 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number (r(2) = 0.91) and total islet number (r(2) = 0.88) and thus increased to r(2) = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method (p < 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method (p < 0.001). The ADIA method also detected small islets between 10 and 50 µm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this technology to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

Oxygenation of the Intraportally Transplanted Pancreatic Islet

PubMed Central

2016-01-01

Intraportal islet transplantation (IT) is not widely utilized as a treatment for type 1 diabetes. Oxygenation of the intraportally transplanted islet has not been studied extensively. We present a diffusion-reaction model that predicts the presence of an anoxic core and a larger partly functional core within intraportally transplanted islets. Four variables were studied: islet diameter, islet fractional viability, external oxygen partial pressure (P) (in surrounding portal blood), and presence or absence of a thrombus on the islet surface. Results indicate that an islet with average size and fractional viability exhibits an anoxic volume fraction (AVF) of 14% and a function loss of 72% at a low external P. Thrombus formation increased AVF to 30% and function loss to 92%, suggesting that the effect of thrombosis may be substantial. External P and islet diameter accounted for the greatest overall impact on AVF and loss of function. At our institutions, large human alloislets (>200 μm diameter) account for ~20% of total islet number but ~70% of total islet volume; since most of the total transplanted islet volume is accounted for by large islets, most of the intraportal islet cells are likely to be anoxic and not fully functional. PMID:27872862

Oxygenation of the Intraportally Transplanted Pancreatic Islet.

PubMed

Suszynski, Thomas M; Avgoustiniatos, Efstathios S; Papas, Klearchos K

2016-01-01

Intraportal islet transplantation (IT) is not widely utilized as a treatment for type 1 diabetes. Oxygenation of the intraportally transplanted islet has not been studied extensively. We present a diffusion-reaction model that predicts the presence of an anoxic core and a larger partly functional core within intraportally transplanted islets. Four variables were studied: islet diameter, islet fractional viability, external oxygen partial pressure ( P ) (in surrounding portal blood), and presence or absence of a thrombus on the islet surface. Results indicate that an islet with average size and fractional viability exhibits an anoxic volume fraction (AVF) of 14% and a function loss of 72% at a low external P . Thrombus formation increased AVF to 30% and function loss to 92%, suggesting that the effect of thrombosis may be substantial. External P and islet diameter accounted for the greatest overall impact on AVF and loss of function. At our institutions, large human alloislets (>200 μ m diameter) account for ~20% of total islet number but ~70% of total islet volume; since most of the total transplanted islet volume is accounted for by large islets, most of the intraportal islet cells are likely to be anoxic and not fully functional.

Antigen recognition in the islets changes with progression of autoimmune islet infiltration

PubMed Central

Lindsay, Robin S.; Corbin, Kaitlin; Mahne, Ashley; Levitt, Bonnie E.; Gebert, Matthew J.; Wigton, Eric J.; Bradley, Brenda J.; Haskins, Kathryn; Jacobelli, Jordan; Tang, Qizhi; Krummel, Matthew F.; Friedman, Rachel S.

2014-01-01

In type 1 diabetes, the pancreatic islets are an important site for therapeutic intervention since immune infiltration of the islets is well established at diagnosis. Therefore, understanding the events that underlie the continued progression of the autoimmune response and islet destruction is critical. Islet infiltration and destruction is an asynchronous process, making it important to analyze the disease process on a single islet basis. To understand how T cell stimulation evolves through the process of islet infiltration we analyzed the dynamics of T cell movement and interactions within individual islets of spontaneously autoimmune non-obese diabetic (NOD) mice. Using both intra-vital and explanted 2-photon islet imaging, we defined a correlation between increased islet infiltration and increased T cell motility. Early T cell arrest was antigen dependent and due, at least in part, to antigen recognition through sustained interactions with CD11c+ antigen presenting cells (APCs). As islet infiltration progressed, T cell motility became antigen-independent, with a loss of T cell arrest and sustained interactions with CD11c+ APCs. These studies suggest that the autoimmune T cell response in the islets may be temporarily dampened during the course of islet infiltration and disease progression. PMID:25505281

Activin receptor-like kinase 5 inhibition reverses impairment of endothelial cell viability by endogenous islet mesenchymal stromal cells.

PubMed

Clarkin, Claire E; King, Aileen J; Dhadda, Paramjeet; Chagastelles, Pedro; Nardi, Nance; Wheeler-Jones, Caroline P; Jones, Peter M

2013-03-01

Following islet transplantation, islet graft revascularization is compromised due to loss of endothelial cells (ECs) during islet culture. TGF-β signaling pathways are essential for vascular homeostasis but their importance for islet EC function is unclear. We have identified a population of multipotent mesenchymal stromal cells (MSCs) within islets and investigated how modulation of TGF-β signaling by these cells influences islet EC viability. Cultured islets exhibited reduced expression of EC markers (VEGFR2, VE-cadherin and CD31), which was associated with diminished but sustained expression of endoglin a marker of both ECs and MSCs. Double fluorescent labeling of islets in situ with the EC marker CD31 disclosed a population of CD31-negative cells which were positive for endoglin. In vitro coculture of microvascular ECs with endoglin-positive, CD31-negative islet MSCs reduced VEGFR2 protein expression, disrupted EC angiogenic behavior, and increased EC detachment. Medium conditioned by islet MSCs significantly decreased EC viability and increased EC caspase 3/7 activity. EC:MSC cocultures showed enhanced Smad2 phosphorylation consistent with altered ALK5 signaling. Pharmacological inhibition of ALK5 activity with SB431542 (SB) improved EC survival upon contact with MSCs, and SB-treated cultured islets retained EC marker expression and sensitivity to exogenous VEGF164 . Thus, endoglin-expressing islet MSCs influence EC ALK5 signaling in vitro, which decreases EC viability, and changes in ALK5 activity in whole cultured islets contribute to islet EC loss. Modifying TGF-β signaling may enable maintenance of islet ECs during islet isolation and thus improve islet graft revascularization post-transplantation. Copyright © 2013 AlphaMed Press.

Engraftment Site and Effectiveness of the Pan-Caspase Inhibitor F573 to Improve Engraftment in Mouse and Human Islet Transplantation in Mice.

PubMed

Pepper, Andrew R; Bruni, Antonio; Pawlick, Rena; Wink, John; Rafiei, Yasmin; Gala-Lopez, Boris; Bral, Mariusz; Abualhassan, Nasser; Kin, Tatsuya; Shapiro, A M James

2017-10-01

Islet transplantation is an effective therapy in type 1 diabetes and recalcitrant hypoglycemia. However, there is an ongoing need to circumvent islet loss posttransplant. We explore herein the potential of the pan-caspase inhibitor F573 to mitigate early apoptosis-mediated islet death within portal and extrahepatic portal sites in mice. Mouse or human islets were cultured in standard media ±100 μM F573 and subsequently assessed for viability and apoptosis via terminal deoxynucleotidyl transferase dUTP nick end labeling staining and caspase-3 activation. Diabetic mice were transplanted with syngeneic islets placed under the kidney capsule (KC) or into the subcutaneous deviceless (DL) site at a marginal islet dose (150 islets), or into the portal vein (PV) at a full dose (500 islets). Human islets were transplanted under the KC of diabetic immunodeficient mice at a marginal dose (500 islet equivalents). Islets were cultured in the presence of F573, and F573 was administered subcutaneously on days 0 to 5 posttransplant. Control mice were transplanted with nontreated islets and were injected with saline. Graft function was measured by nonfasting blood glucose and glucose tolerance testing. F573 markedly reduced human and mouse islet apoptosis after in vitro culture (P < 0.05 and P < 0.05, respectively). Furthermore, F573 improved human islet function when transplanted under the KC (P < 0.05); whereas F573 did not enhance murine islet marginal KC transplants. Conversely, F573 significantly improved mouse islet engraftment in the PV and DL site (P < 0.05 and P < 0.05, respectively). The pan-caspase inhibitor F573 markedly reduces human and mouse islet apoptosis and improves engraftment most effectively in the portal and DL subcutaneous sites.

A replacement for islet equivalents with improved reliability and validity.

PubMed

Huang, Han-Hung; Ramachandran, Karthik; Stehno-Bittel, Lisa

2013-10-01

Islet equivalent (IE), the standard estimate of isolated islet volume, is an essential measure to determine the amount of transplanted islet tissue in the clinic and is used in research laboratories to normalize results, yet it is based on the false assumption that all islets are spherical. Here, we developed and tested a new easy-to-use method to quantify islet volume with greater accuracy. Isolated rat islets were dissociated into single cells, and the total cell number per islet was determined by using computer-assisted cytometry. Based on the cell number per islet, we created a regression model to convert islet diameter to cell number with a high R2 value (0.8) and good validity and reliability with the same model applicable to young and old rats and males or females. Conventional IE measurements overestimated the tissue volume of islets. To compare results obtained using IE or our new method, we compared Glut2 protein levels determined by Western Blot and proinsulin content via ELISA between small (diameter≤100 μm) and large (diameter≥200 μm) islets. When normalized by IE, large islets showed significantly lower Glut2 level and proinsulin content. However, when normalized by cell number, large and small islets had no difference in Glut2 levels, but large islets contained more proinsulin. In conclusion, normalizing islet volume by IE overestimated the tissue volume, which may lead to erroneous results. Normalizing by cell number is a more accurate method to quantify tissue amounts used in islet transplantation and research.

Pharmacological strategies for protection of extrahepatic islet transplantation.

PubMed

Omori, K; Komatsu, H; Rawson, J; Mullen, Y

2015-06-01

The safety and effectiveness of islet transplantation has been proven through world-wide trials. However, acute and chronic islet loss has hindered the ultimate objective of becoming a widely used treatment option for type 1 diabetes. A large islet loss is attributed, in part, to the liver being a less-than-optimal site for transplantation. Over half of the transplanted islets are destroyed shortly after transplantation due to direct exposure to blood and non-specific inflammation. Successfully engrafted islets are continuously exposed to the liver micro-environment, a unique immune system, low oxygen tension, toxins and high glucose, which is toxic to islets, leading to premature islet dysfunction/death. Investigations have continued to search for alternate sites to transplant islets that provide a better environment for prolonged function and survival. This article gathers courses and conditions that lead to islet loss, from organ procurement through islet transplantation, with special emphasis on hypoxia, oxidative stress, and antigen non-specific inflammation, and reviews strategies using pharmacological agents that have shown effectiveness in protecting islets, including a new treatment approach utilizing siRNA. Pharmacological agents that support islet survival and promote β-cell proliferation are also included. Treatment of donor pancreata and/or islets with these agents should increase the effectiveness of islets transplanted into extrahepatic sites. Furthermore, the development of methods designed to release these agents over an extended period, will further increase their efficacy. This requires the combined efforts of both islet transplant biologists and bioengineers.

Fission of pancreatic islets during postnatal growth of the mouse

PubMed Central

Seymour, Philip A; Bennett, William R; Slack, Jonathan M W

2004-01-01

A cell composition analysis was made of the pancreatic islets in postnatal H253 mice. This line has a lacZ insertion on the X chromosome so that in female hemizygotes 50% of cells should be positive for β-galactosidase and 50% negative. Immediately after birth, the islets were of a heterogeneous cell composition. However, by 4 weeks some islets have become homogeneous. This suggests that islets progress towards monoclonality in a similar way to the intestinal crypts and stomach gastric glands. Pancreatic islets may therefore represent ‘structural proliferative units’ in the overall histological organization of the pancreas. Reduction of genetic heterogeneity might arise from cell turnover, fission of islets or both. Analysis of the cell composition of the X-inactivation mosaic mice also provides the first clear evidence for islet fission in pancreatic development. Irregularly shaped islets resembling dumb-bells, with a characteristic neck of α-cells, were observed with decreasing frequency with increasing age. Three-dimensional reconstruction confirmed their resemblance to conjoined islets. The cell composition analysis showed: (1) the relatedness of the two sides of a dumb-bell islet is significantly higher than between two non-dumb-bell islets and (2) the relatedness of two randomly selected islets decreases as the distance between them increases. This suggests that dumb-bell islets are in a state of fission rather than fusion, and that islet fission is a mode of islet production in the postnatal pancreas. PMID:15032917

Autologous islet transplantation: challenges and lessons.

PubMed

Dunn, Ty B; Wilhelm, Joshua J; Bellin, Melena D; Pruett, Timothy L

2017-08-01

Human islet isolation and autotransplantation [autologous islet transplant (AUTX)] is performed to prevent or ameliorate brittle diabetes after total pancreatectomy performed for benign disease. The success or failure of the transplant can be associated with a profound impact on the individual's quality of life and even survival. AUTX offers unique insights into the effects of pancreas quality, islet number, isolation technique and alternate site engraftment on transplant efficacy. Herein, we review islet isolation with a focus on potential pathways to further optimize the endocrine outcome of AUTX, and compare and contrast differences in islet processing for AUTX and allotransplantation (allogeneic islet transplant). New knowledge of human islet biology and issues surrounding the engraftment process offer opportunities for innovative approaches toward optimizing islet cell transplantation. Improving the rate and durability of insulin independence in the often-times marginal dose model of AUTX may provide new insight toward improving the efficiency and durability of single donor islet (allogeneic islet transplant).

Feasibility of islet magnetic resonance imaging using ferumoxytol in intraportal islet transplantation.

PubMed

Jin, Sang-Man; Oh, Seung-Hoon; Oh, Bae Jun; Shim, Wooyoung; Choi, Jin Myung; Yoo, Dongkyeom; Hwang, Yong Hwa; Lee, Jung Hee; Lee, Dong Yun; Kim, Jae Hyeon

2015-06-01

There is a clinical need for an alternative labeling agent for magnetic resonance imaging (MRI) in islet transplantation. We aimed to evaluate the feasibility of islet MRI using ferumoxytol, which is the only clinically-available ultrasmall superparamagnetic iron oxide. We compared islet function and viability of control islets and islets labeled with ferumoxytol and/or a heparin-protamine complex (HPF). Efficacy of ferumoxytol labeling was assessed in both ex vivo and in vivo models. Labeling for 48 h with HPF, but not up to 800 μg/mL ferumoxytol, deranged ex vivo islet viability and function. The T2∗ relaxation time was optimal when islets were labeled with 800 μg/mL of ferumoxytol for 48 h. Prussian blue stain, iron content assay, transmission electron microscopy (TEM) supported internalization of ferumoxytol particles. However, the labeling intensity in the ex vivo MRI of islets labeled with ferumoxytol was much weaker than that of islets labeled with ferucarbotran. In syngeneic intraportal islet transplantation, there was a correlation between the total area of visualized islets and the transplanted islet mass. In conclusion, islet MRI using ferumoxytol was feasible in terms of in vitro and in vivo efficacy and safety. However, the weak labeling efficacy is still a hurdle for the clinical application. Copyright © 2015 Elsevier Ltd. All rights reserved.

Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

PubMed

Oura, Tetsu; Ko, Dicken S C; Boskovic, Svjetlan; O'Neil, John J; Chipashvili, Vaja; Koulmanda, Maria; Hotta, Kiyohiko; Kawai, Kento; Nadazdin, Ognjenka; Smith, R Neal; Cosimi, A B; Kawai, Tatsuo

2016-01-01

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Pleckstrin homology-like domain family A, member 3 (PHLDA3) deficiency improves islets engraftment through the suppression of hypoxic damage

PubMed Central

Yamaguchi, Yohko; Chen, Yu; Shimoda, Masayuki; Yoshimatsu, Gumpei; Unno, Michiaki; Sumi, Shoichiro; Ohki, Rieko

2017-01-01

Islet transplantation is a useful cell replacement therapy that can restore the glycometabolic function of severe diabetic patients. It is known that many transplanted islets failed to engraft, and thus, new approaches for overcoming graft loss that may improve the outcome of future clinical islet transplantations are necessary. Pleckstrin homology-like domain family A, member 3 (PHLDA3) is a known suppressor of neuroendocrine tumorigenicity, yet deficiency of this gene increases islet proliferation, prevents islet apoptosis, and improves their insulin-releasing function without causing tumors. In this study, we examined the potential use of PHLDA3-deficient islets in transplantation. We observed that: 1) transplanting PHLDA3-deficient islets into diabetic mice significantly improved their glycometabolic condition, 2) the improved engraftment of PHLDA3-deficient islets resulted from increased cell survival during early transplantation, and 3) Akt activity was elevated in PHLDA3-deficient islets, especially under hypoxic conditions. Thus, we determined that PHLDA3-deficient islets are more resistant against stresses induced by islet isolation and transplantation. We conclude that use of islets with suppressed PHLDA3 expression could be a novel and promising treatment for improving engraftment and consequent glycemic control in islet transplantation. PMID:29121094

Isolated human islets require hyperoxia to maintain islet mass, metabolism, and function.

PubMed

Komatsu, Hirotake; Kang, Dongyang; Medrano, Leonard; Barriga, Alyssa; Mendez, Daniel; Rawson, Jeffrey; Omori, Keiko; Ferreri, Kevin; Tai, Yu-Chong; Kandeel, Fouad; Mullen, Yoko

2016-02-12

Pancreatic islet transplantation has been recognized as an effective treatment for Type 1 diabetes; however, there is still plenty of room to improve transplantation efficiency. Because islets are metabolically active they require high oxygen to survive; thus hypoxia after transplant is one of the major causes of graft failure. Knowing the optimal oxygen tension for isolated islets would allow a transplant team to provide the best oxygen environment during pre- and post-transplant periods. To address this issue and begin to establish empirically determined guidelines for islet maintenance, we exposed in vitro cultured islets to different partial oxygen pressures (pO2) and assessed changes in islet volume, viability, metabolism, and function. Human islets were cultured for 7 days in different pO2 media corresponding to hypoxia (90 mmHg), normoxia (160 mmHg), and hyerpoxia (270 or 350 mmHg). Compared to normoxia and hypoxia, hyperoxia alleviated the loss of islet volume, maintaining higher islet viability and metabolism as measured by oxygen consumption and glucose-stimulated insulin secretion responses. We predict that maintaining pre- and post-transplanted islets in a hyperoxic environment will alleviate islet volume loss and maintain islet quality thereby improving transplant outcomes. Copyright © 2016 Elsevier Inc. All rights reserved.

A novel redox-active metalloporphyrin reduces reactive oxygen species and inflammatory markers but does not improve marginal mass engraftment in a murine donation after circulatory death islet transplantation model

PubMed Central

Bruni, Antonio; Pepper, Andrew R.; Gala-Lopez, Boris; Pawlick, Rena; Abualhassan, Nasser; Crapo, James D.; Piganelli, Jon D.; Shapiro, A. M. James

2016-01-01

ABSTRACT Islet transplantation is a highly effective treatment for stabilizing glycemic control for select patients with type-1 diabetes. Despite improvements to clinical transplantation, single-donor transplant success has been hard to achieve routinely, necessitating increasing demands on viable organ availability. Donation after circulatory death (DCD) may be an alternative option to increase organ availability however, these organs tend to be more compromised. The use of metalloporphyrin anti-inflammatory and antioxidant (MnP) compounds previously demonstrated improved in vivo islet function in preclinical islet transplantation. However, the administration of MnP (BMX-001) in a DCD islet isolation and transplantation model has yet to be established. In this study, murine donors were subjected to a 15-min warm ischemic (WI) period prior to isolation and culture with or without MnP. Subsequent to one-hour culture, islets were assessed for in vitro viability and in vivo function. A 15-minute WI period significantly reduced islet yield, regardless of MnP-treatment relative to yields from standard isolation. MnP-treated islets did not improve islet viability compared to DCD islets alone. MnP-treatment did significantly reduce the presence of extracellular reactive oxygen species (ROS) (p < 0 .05). Marginal, syngeneic islets (200 islets) transplanted under the renal capsule exhibited similar in vivo outcomes regardless of WI or MnP-treatment. DCD islet grafts harvested 7 d post-transplant exhibited sustained TNF-α and IL-10, while MnP-treated islet-bearing grafts demonstrated reduced IL-10 levels. Taken together, 15-minute WI in murine islet isolation significantly impairs islet yield. DCD islets do indeed demonstrate in vivo function, though MnP therapy was unable to improve viability and engraftment outcomes. PMID:27220256

Assessing the effect of immunosuppression on engraftment of pancreatic islets

PubMed Central

Vallabhajosyula, Prashanth; Hirakata, Atsushi; Shimizu, Akira; Okumi, Masayoshi; Tchipashvili, Vaja; Hong, Hanzhou; Yamada, Kazuhiko; Sachs, David H.

2013-01-01

Objective In addition to ischemia and immunologic factors, immunosuppressive drugs have been suggested as a possible contributing factor to the loss of functional islets following allogeneic islet cell transplantation. Using our previously described islet-kidney transplantation model in miniature swine, we studied whether an islet toxic triple-drug immunosuppressive regimen (cyclosporine + azathioprine + prednisone) affects the islet engraftment process and thus long-term islet function. Design and Methods Donor animals underwent partial pancreatectomy, autologous islet preparation and injection of these islets under the autologous kidney capsule to prepare an islet-kidney (IK). Experimental animals received daily triple drug immunosuppression during the islet engraftment period. Control animals did not receive any immunosuppression during this period. Four to eight weeks later, these engrafted IK were transplanted across a minor histocompatibility mismatched barrier into pancreatectomized, nephrectomized recipient animals at an islet dose of ~ 4500 islet equivalents (IE)/kg recipient weight. Cyclosporine was administered for 12 days to the recipients to induce tolerance of the IK grafts and the animals were followed long-term. Results Diabetes was corrected by IK transplantation in all pancreatectomized recipients on both the control (n=3) and the experimental (n=4) arms of the study and all animals showed normal glucose regulation over the follow-up period. Intravenous glucose tolerance tests performed at 1, 2, > 3 months post-IK transplant showed essentially equivalent glycemic control in both control and experimental animals. Conclusion In this pre-clinical, in vivo large animal model of islet transplantation, the effect of triple drug immunosuppression on islet function does not negatively affect islet engraftment, as assessed by the long-term function of engrafted islets. PMID:23883972

A novel redox-active metalloporphyrin reduces reactive oxygen species and inflammatory markers but does not improve marginal mass engraftment in a murine donation after circulatory death islet transplantation model.

PubMed

Bruni, Antonio; Pepper, Andrew R; Gala-Lopez, Boris; Pawlick, Rena; Abualhassan, Nasser; Crapo, James D; Piganelli, Jon D; Shapiro, A M James

2016-07-03

Islet transplantation is a highly effective treatment for stabilizing glycemic control for select patients with type-1 diabetes. Despite improvements to clinical transplantation, single-donor transplant success has been hard to achieve routinely, necessitating increasing demands on viable organ availability. Donation after circulatory death (DCD) may be an alternative option to increase organ availability however, these organs tend to be more compromised. The use of metalloporphyrin anti-inflammatory and antioxidant (MnP) compounds previously demonstrated improved in vivo islet function in preclinical islet transplantation. However, the administration of MnP (BMX-001) in a DCD islet isolation and transplantation model has yet to be established. In this study, murine donors were subjected to a 15-min warm ischemic (WI) period prior to isolation and culture with or without MnP. Subsequent to one-hour culture, islets were assessed for in vitro viability and in vivo function. A 15-minute WI period significantly reduced islet yield, regardless of MnP-treatment relative to yields from standard isolation. MnP-treated islets did not improve islet viability compared to DCD islets alone. MnP-treatment did significantly reduce the presence of extracellular reactive oxygen species (ROS) (p < 0 .05). Marginal, syngeneic islets (200 islets) transplanted under the renal capsule exhibited similar in vivo outcomes regardless of WI or MnP-treatment. DCD islet grafts harvested 7 d post-transplant exhibited sustained TNF-α and IL-10, while MnP-treated islet-bearing grafts demonstrated reduced IL-10 levels. Taken together, 15-minute WI in murine islet isolation significantly impairs islet yield. DCD islets do indeed demonstrate in vivo function, though MnP therapy was unable to improve viability and engraftment outcomes.

Islet Transplantation without Borders Enabling islet transplantation in Greece with international collaboration and innovative technology

PubMed Central

Papas, Klearchos K; Karatzas, Theodore; Berney, Thierry; Minor, Thomas; Pappas, Paris; Pattou, François; Shaw, James; Toso, Christian; Schuurman, Henk-Jan

2012-01-01

Recently, initiatives have been undertaken to establish an islet transplantation program in Athens, Greece. A major hurtle is the high cost associated with the establishment and maintenance of a clinical-grade islet manufacturing center. A collaboration was established with the University Hospitals of Geneva, Switzerland, to enable remote islet cell manufacturing with an established and validated fully operational team. However, remote islet manufacturing requires shipment of the pancreas from the procurement to the islet manufacturing site (in this case from anywhere in Greece to Geneva) and then shipment of the islets from the manufacturing site to the transplant site (from Geneva to Athens). To address challenges related to cold ischemia time of the pancreas and shipment time of islets, a collaboration was initiated with the University of Arizona, Tucson, USA. An international workshop was held in Athens, December 2011, to mark the start of this collaborative project. Experts in the field presented in three main sessions: [1] Islet transplantation: state-of-the-art, and the “network approach”; [2] Technical aspects of clinical islet transplantation and outcomes; and [3] Islet manufacturing – from the donated pancreas to the islet product. This manuscript presents a summary of the workshop. PMID:23330863

Enhanced expression of VEGF-A in β cells increases endothelial cell number but impairs islet morphogenesis and β cell proliferation

PubMed Central

Cai, Qing; Brissova, Marcela; Reinert, Rachel B.; Pan, Fong Cheng; Brahmachary, Priyanka; Jeansson, Marie; Shostak, Alena; Radhika, Aramandla; Poffenberger, Greg; Quaggin, Susan E.; Jerome, W. Gray; Dumont, Daniel J.; Powers, Alvin C.

2012-01-01

There is a reciprocal interaction between pancreatic islet cells and vascular endothelial cells (EC) in which EC-derived signals promote islet cell differentiation and islet development while islet cell-derived angiogenic factors promote EC recruitment and extensive islet vascularization. To examine the role of angiogenic factors in the coordinated development of islets and their associated vessels, we used a “tet-on” inducible system (mice expressing rat insulin promoter-reverse tetracycline activator transgene and a tet-operon-angiogenic factor transgene) to increase the β cell production of vascular endothelial growth factor-A (VEGF-A), angiopoietin-1 (Ang1), or angiopoietin-2 (Ang2) during islet cell differentiation and islet development. In VEGF-A overexpressing embryos, ECs began to accumulate around epithelial tubes residing in the central region of the developing pancreas (associated with endocrine cells) as early as embryonic day 12.5 (E12.5) and increased dramatically by E16.5. While α and β cells formed islet cell clusters in control embryos at E16.5, the increased EC population perturbed endocrine cell differentiation and islet cell clustering in VEGF-A overexpressing embryos. With continued overexpression of VEGF-A, α and β cells became scattered, remained adjacent to ductal structures, and never coalesced into islets, resulting in a reduction in β cell proliferation and β cell mass at postnatal day 1. A similar impact on islet morphology was observed when VEGF-A was overexpressed in β cells during the postnatal period. In contrast, increased expression of Ang1 or Ang2 in β cells in developing or adult islets did not alter islet differentiation, development, or morphology, but altered islet EC ultrastructure. These data indicate that 1) increased EC number does not promote, but actually impairs β cell proliferation and islet formation; 2) the level of VEGF-A production by islet endocrine cells is critical for islet vascularization during development and postnatally; 3) Angiopoietin-Tie2 signaling in endothelial cells does not have a crucial role in the development or maintenance of islet vascularization. PMID:22546694

Adverse effect on syngeneic islet transplantation by transgenic coexpression of decoy receptor 3 and heme oxygenase-1 in the islet of NOD mice.

PubMed

Huang, S-H; Lin, G-J; Chien, M-W; Chu, C-H; Yu, J-C; Chen, T-W; Hueng, D-Y; Liu, Y-L; Sytwu, H-K

2013-03-01

Decoy receptor 3 (DcR3) blocks both Fas ligand- and LIGHT-induced pancreatic β-cell damage in autoimmune diabetes. Heme oxygenase 1 (HO-1) possesses antiapoptotic, anti-inflammatory, and antioxidative effects that protect cells against various forms of attack by the immune system. Previously, we have demonstrated that transgenic islets overexpressing DcR3 or murine HO-1 (mHO-1) exhibit longer survival times than nontransgenic islets in syngeneic islet transplantation. In this study, we evaluated whether DcR3 and mHO-1 double-transgenic islets of NOD mice could provide better protective effects and achieve longer islet graft survival than DcR3 or mHO-1 single-transgenic islets after islet transplantation. We generated DcR3 and mHO-1 double-transgenic NOD mice that specifically overexpress DcR3 and HO-1 in islets. Seven hundred islets isolated from double-transgenic, single-transgenic, or nontransgenic NOD mice were syngeneically transplanted into the kidney capsules of newly diabetic female recipients. Unexpectedly, there was no significant difference in the survival time between double-transgenic or nontransgenic NOD islet grafts, and the survival times of double-transgenic NOD islet grafts were even shorter than those of DcR3 or mHO-1 single-transgenic islets. Our data indicate that transplantation of double-transgenic islets that coexpress HO-1 and DcR3 did not result in a better outcome. On the contrary, this strategy even caused an adverse effect in syngeneic islet transplantation. Copyright © 2013 Elsevier Inc. All rights reserved.

Quantitative Differential Expression Analysis Reveals Mir-7 As Major Islet MicroRNA

PubMed Central

Bravo-Egana, Valia; Rosero, Samuel; Molano, R. Damaris; Pileggi, Antonello; Ricordi, Camillo; Domínguez-Bendala, Juan; Pastori, Ricardo L.

2008-01-01

MicroRNAs (miRNAs) are non-coding gene products that regulate gene expression through specific binding to target mRNAs. Cell-specific patterns of miRNAs are associated with the acquisition and maintenance of a given phenotype, such as endocrine pancreas (islets). We hypothesized that a subset of miRNAs could be differentially expressed in the islets. Using miRNA microarray technology and quantitative RT-PCR we identified a subset of miRNAs that are the most differentially expressed islet miRNAs (ratio islet/acinar >150-fold), mir-7 being the most abundant. A similarly high ratio for mir-7 was observed in human islets. The ratio islet/acinar for mir-375, a previously described islet miRNA, was <10, and is 2.5X more abundant in the islets than mir-7. Therefore, we conclude that mir-7 is the most abundant endocrine miRNA in islets while mir-375 is the most abundant intra-islet miRNA. Our results may offer new insights into regulatory pathways of islet gene expression. PMID:18086561

[Islet isolation outcome is influenced by pancreas preparation method].

PubMed

Pokrywczyńska, Marta; Drewa, Tomasz; Cieślak, Zaneta

2008-09-01

Pancreatic islet transplantation is a treatment method for type I diabetes. Its outcome is influenced by numerous factors, islet quantity and function being important ones of them. was to estimate the influence of pancreas preparation method on the outcome of islet isolation in rat. 6 pancreata harvested from Lewis rats were used in this research. Pancreatic duct was cannulated and pancreas was injected with 1 mg/ml collagenase P solution (Sigma) and then excised. After cutting into smaller fragments, it was digested in collagenase P solution for 15-20 min. Enzyme activity was then stopped by adding dilution medium. Heterogenous cell suspension was centrifuged in density gradient (Gradisol) to isolate islets. Pancreatic islets were collected and islet equivalent was calculated. Islet purity degree was estimated as islet cells to all cells, including exocrine, ratio. Islet viability was estimated using propidium iodide and fluorescein diacetate staining. Photographic documentation was made. Proper islet morphology, highest number and viability was obtained when pancreas was excised properly (isolation 3 and 4). Pancreas preparation method is one of which influences on islet isolation outcome.

Vascular reactivity in arterioles from normal and alloxan-diabetic mice: studies on single perfused islets.

PubMed

Lai, En Yin; Jansson, Leif; Patzak, Andreas; Persson, A Erik G

2007-01-01

Pancreatic islets possess an autonomous mechanism of blood flow regulation, independent of that of the exocrine pancreas. To study islet vascular regulation without confounding effects of the exocrine blood vessels, we have developed a technique enabling us to isolate single pancreatic islets and then to perfuse them using their endogenous vasculature for distribution of the medium. This made it possible to directly study the vascular reactivity of islet arterioles to different substances. We confirmed that control of islet blood flow is mainly located at the precapillary level. As expected, administration of angiotensin II and l-nitro-arginine methyl ester contracted islet arterioles, whereas nitric oxide and adenosine dilated them. d-glucose, the main insulin secretagogue, had a selective dilating effect on smooth muscle in islet arterioles but not in glomerular afferent arterioles. The response to glucose was amplified in islet arterioles from diabetic animals, indicating enhanced islet blood perfusion in diabetes. This newly developed technique for perfusing isolated pancreatic islets will provide new insights into islet perfusion control and its possible contributions to the pathogenesis of type 2 diabetes.

Infected Peripheral Blood Mononuclear Cells Transmit Latent Varicella Zoster Virus Infection to the Guinea Pig Enteric Nervous System

PubMed Central

Gan, Lin; Wang, Mingli; Chen, Jason J.; Gershon, Michael D.; Gershon, Anne A.

2014-01-01

Latent wild-type (WT) and vaccine (vOka) varicella-zoster virus (VZV) are found in the human enteric nervous system (ENS). VZV also infects guinea pig enteric neurons in vitro, establishes latency and can be reactivated. We therefore determined whether lymphocytes infected in vitro with VZV secrete infectious virions and can transfer infection in vivo to the ENS of recipient guinea pigs. T lymphocytes (CD3-immunoreactive) were preferentially infected following co-culture of guinea pig or human peripheral blood mononuclear cells with VZV-infected HELF. VZV proliferated in the infected T cells and expressed immediate early and late VZV genes. Electron microscopy confirmed that VZV-infected T cells produced encapsulated virions. Extracellular virus, however, was pleomorphic, suggesting degradation occurred prior to release, which was confirmed by the failure of VZV-infected T cells to secrete infectious virions. Intravenous injection of WT- or vOka-infected PBMCs, nevertheless, transmitted VZV to recipient animals (guinea pig > human lymphocytes). Two days post-inoculation, lung and liver, but not gut, contained DNA and transcripts encoding ORFs 4, 40, 66 and 67. Twenty-eight days after infection, gut contained DNA and transcripts encoding ORFs 4 and 66 but neither DNA nor transcripts could any longer be found in lung or liver. In situ hybridization revealed VZV DNA in enteric neurons, which also expressed ORF63p (but not ORF68p) immunoreactivity. Observations suggest that VZV infects T cells, which can transfer VZV to and establish latency in enteric neurons in vivo. Guinea pigs may be useful for studies of VZV pathogenesis in the ENS. PMID:24965252

A novel subcutaneous site of islet transplantation superior to the liver.

PubMed

Yasunami, Yohichi; Nakafusa, Yuki; Nitta, Naoyoshi; Nakamura, Masafumi; Goto, Masafumi; Ono, Junko; Taniguchi, Masaru

2018-03-08

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 2-3 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 STZ-induced diabetes in mice. Importantly, transplanted islets can be visualized by CT and are easily retrievable, and allograft rejection is preventable by blockade of co-stimulatory 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.

Macroporous biohybrid cryogels for co-housing pancreatic islets with mesenchymal stromal cells.

PubMed

Borg, Danielle J; Welzel, Petra B; Grimmer, Milauscha; Friedrichs, Jens; Weigelt, Marc; Wilhelm, Carmen; Prewitz, Marina; Stißel, Aline; Hommel, Angela; Kurth, Thomas; Freudenberg, Uwe; Bonifacio, Ezio; Werner, Carsten

2016-10-15

Intrahepatic transplantation of allogeneic pancreatic islets offers a promising therapy for type 1 diabetes. However, long-term insulin independency is often not achieved due to severe islet loss shortly after transplantation. To improve islet survival and function, extrahepatic biomaterial-assisted transplantation of pancreatic islets to alternative sites has been suggested. Herein, we present macroporous, star-shaped poly(ethylene glycol) (starPEG)-heparin cryogel scaffolds, covalently modified with adhesion peptides, for the housing of pancreatic islets in three-dimensional (3D) co-culture with adherent mesenchymal stromal cells (MSC) as accessory cells. The implantable biohybrid scaffolds provide efficient transport properties, mechanical protection, and a supportive extracellular environment as a desirable niche for the islets. MSC colonized the cryogel scaffolds and produced extracellular matrix proteins that are important components of the natural islet microenvironment known to facilitate matrix-cell interactions and to prevent cellular stress. Islets survived the seeding procedure into the cryogel scaffolds and secreted insulin after glucose stimulation in vitro. In a rodent model, intact islets and MSC could be visualized within the scaffolds seven days after subcutaneous transplantation. Overall, this demonstrates the potential of customized macroporous starPEG-heparin cryogel scaffolds in combination with MSC to serve as a multifunctional islet supportive carrier for transplantation applications. Diabetes results in the insufficient production of insulin by the pancreatic β-cells in the islets of Langerhans. Transplantation of pancreatic islets offers valuable options for treating the disease; however, many transplanted islets often do not survive the transplantation or die shortly thereafter. Co-transplanted, supporting cells and biomaterials can be instrumental for improving islet survival, function and protection from the immune system. In the present study, islet supportive hydrogel sponges were explored for the co-transplantation of islets and mesenchymal stromal cells. Survival and continued function of the supported islets were demonstrated in vitro. The in vivo feasibility of the approach was shown by transplantation in a mouse model. Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

AUTONOMIC AXONS IN THE HUMAN ENDOCRINE PANCREAS SHOW UNIQUE INNERVATION PATTERNS

PubMed Central

Rodriguez-Diaz, Rayner; Abdulreda, Midhat H.; Formoso, Alexander L.; Gans, Itai; Ricordi, Camillo; Berggren, Per-Olof; Caicedo, Alejandro

2011-01-01

SUMMARY The autonomic nervous system regulates hormone secretion from the endocrine pancreas, the islets of Langerhans, and thus impacts glucose metabolism. The parasympathetic and sympathetic nerves innervate the pancreatic islet, but the precise innervation patterns are not known, particularly in human islets. Here we demonstrate that the innervation of human islets is different from that of mouse islets and that it does not conform to existing models of autonomic control of islet function. By visualizing axons in three dimensions and quantifying axonal densities and contacts within pancreatic islets, we found that, in contrast to mouse endocrine cells, human endocrine cells are sparsely contacted by autonomic axons. Few parasympathetic cholinergic axons penetrate the human islet and the invading sympathetic fibers preferentially innervate smooth muscle cells of blood vessels located within the islet. Thus, rather than modulating endocrine cell function directly, sympathetic nerves may regulate hormone secretion in human islets by controlling local blood flow or by acting on islet regions located downstream. PMID:21723503

Improving pancreatic islet in vitro functionality and transplantation efficiency by using heparin mimetic peptide nanofiber gels.

PubMed

Uzunalli, Gozde; Tumtas, Yasin; Delibasi, Tuncay; Yasa, Oncay; Mercan, Sercan; Guler, Mustafa O; Tekinay, Ayse B

2015-08-01

Pancreatic islet transplantation is a promising treatment for type 1 diabetes. However, viability and functionality of the islets after transplantation are limited due to loss of integrity and destruction of blood vessel networks. Thus, it is important to provide a proper mechanically and biologically supportive environment for enhancing both in vitro islet culture and transplantation efficiency. Here, we demonstrate that heparin mimetic peptide amphiphile (HM-PA) nanofibrous network is a promising platform for these purposes. The islets cultured with peptide nanofiber gel containing growth factors exhibited a similar glucose stimulation index as that of the freshly isolated islets even after 7 days. After transplantation of islets to STZ-induced diabetic rats, 28 day-long monitoring displayed that islets that were transplanted in HM-PA nanofiber gels maintained better blood glucose levels at normal levels compared to the only islet transplantation group. In addition, intraperitoneal glucose tolerance test revealed that animals that were transplanted with islets within peptide gels showed a similar pattern with the healthy control group. Histological assessment showed that islets transplanted within peptide nanofiber gels demonstrated better islet integrity due to increased blood vessel density. This work demonstrates that using the HM-PA nanofiber gel platform enhances the islets function and islet transplantation efficiency both in vitro and in vivo. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Adipose stem cells from chronic pancreatitis patients improve mouse and human islet survival and function.

PubMed

Song, Lili; Sun, Zhen; Kim, Do-Sung; Gou, Wenyu; Strange, Charlie; Dong, Huansheng; Cui, Wanxing; Gilkeson, Gary; Morgan, Katherine A; Adams, David B; Wang, Hongjun

2017-08-30

Chronic pancreatitis has surgical options including total pancreatectomy to control pain. To avoid surgical diabetes, the explanted pancreas can have islets harvested and transplanted. Immediately following total pancreatectomy with islet autotransplantation (TP-IAT), many islet cells die due to isolation and transplantation stresses. The percentage of patients remaining insulin free after TP-IAT is therefore low. We determined whether cotransplantation of adipose-derived mesenchymal stem cells (ASCs) from chronic pancreatitis patients (CP-ASCs) would protect islets after transplantation. In a marginal mass islet transplantation model, islets from C57BL/6 mice were cotransplanted with CP-ASCs into syngeneic streptozotocin-treated diabetic mice. Treatment response was defined by the percentage of recipients reaching normoglycemia, and by the area under the curve for glucose and c-peptide in a glucose tolerance test. Macrophage infiltration, β-cell apoptosis, and islet graft vasculature were measured in transplanted islet grafts by immunohistochemistry. mRNA expression profiling of 84 apoptosis-related genes in islet grafts transplanted alone or with CP-ASCs was measured by the RT 2 Profiler™ Apoptosis PCR Array. The impact of insulin-like growth factor-1 (IGF-1) on islet apoptosis was determined in islets stimulated with cytokines (IL-1β and IFN-γ) in the presence and absence of CP-ASC conditioned medium. CP-ASC-treated mice were more often normoglycemic compared to mice receiving islets alone. ASC cotransplantation reduced macrophage infiltration, β-cell death, suppressed expression of TNF-α and Bcl-2 modifying factor (BMF), and upregulated expressions of IGF-1 and TNF Receptor Superfamily Member 11b (TNFRSF11B) in islet grafts. Islets cultured in conditioned medium from CP-ASCs showed reduced cell death. This protective effect was diminished when IGF-1 was blocked in the conditioned medium by the anti-IGF-1 antibody. Cotransplantation of islets with ASCs from the adipose of chronic pancreatitis patients improved islet survival and islet function after transplantation. The effects are in part mediated by paracrine secretion of IGF-1, suppression of inflammation, and promotion of angiogenesis. ASCs from chronic pancreatitis patients have the potential to be used as a synergistic therapy to enhance the efficacy of islet transplantation following pancreatectomy.

Kidney versus Islet Allograft Survival after Induction of Mixed Chimerism with Combined Donor Bone Marrow Transplantation

PubMed Central

Oura, Tetsu; Ko, Dicken S.C.; Boskovic, Svjetlan; O'Neil, John J.; Chipashvili, Vaja; Koulmanda, Maria; Hotta, Kiyohiko; Kawai, Kento; Nadazdin, Ognjenka; Smith, R. Neal; Cosimi, A. B.; Kawai, Tatsuo

2016-01-01

Background We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC-mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. Methods A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that includes low dose total body and thymic irradiation, horse ATG (Atgam), six doses of anti-CD154 monoclonal antibody (mAb) and a one month course of cyclosporine (CyA) (Islet-A). In Islet-B, anti-CD8 mAb was administered in place of CyA. In Islet-C, two recipients were treated with Islet-B but without Atgam. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and bone marrow transplantation (Kidney-A) following the same conditioning regimen used in Islet-A. Results The majority of Kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned Islet/BM recipients (Islet-A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to calcineurin inhibitor (CNI) toxicity, three recipients were treated with anti-CD8 mAb in place of CNI. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet-C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Conclusion Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CNI-free regimen that includes anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance. PMID:26337731

Facile mechanical shaking method is an improved isolation approach for islet preparation and transplantation.

PubMed

Yin, Nina; Chen, Tao; Yu, Yuling; Han, Yongming; Yan, Fei; Zheng, Zhou; Chen, Zebin

2016-12-01

Successful islet isolation is crucial for islet transplantation and cell treatment for type 1 diabetes. Current isolation methods are able to obtain 500-1,000 islets per rat, which results in a waste of ≥50% of total islets. In the present study, a facile mechanical shaking method for improving islet yield (up to 1,500 per rat) was developed and summarized, which was demonstrated to be more effective than the existing well-established stationary method. The present results showed that isolated islets have a maximum yield of 1,326±152 when shaking for 15 min for the fully-cannulated pancreas. For both fully-cannulated and half-cannulated pancreas in the presence of rat DNAse inhibitor, the optimal shaking time was amended to 20 min with a further increased yield of 1,344±134 and 1,286±124 islets, respectively. Furthermore, the majority of the isolated islets were morphologically intact with a well-defined surface and almost no central necrotic zone, which suggested that the condition of islets obtained via the mechanical shaking method was consistent with the stationary method. Islet size distribution was also calculated and it was demonstrated that islets from the stationary method exhibited the same size distribution as the non-cannulated group, which had more larger islets than the fully-cannulated and half-cannulated groups isolated via the shaking method. In addition, the results of glucose challenge showed that the refraction index of each group was >2.5, which indicated the well-preserved function of isolated islets. Furthermore, the transplanted islets exhibited a therapeutic effect after 1 day of transplantation; however, they failed to control blood glucose levels after ~7 days of transplantation. In conclusion, these results demonstrated that the facile mechanical shaking method may markedly improve the yield of rat islet isolation, and in vitro and in vivo investigation demonstrated the well-preserved function of isolated islets in the control of blood glucose. Therefore, the facile mechanical shaking method may be an alternative improved procedure to obtain higher islet yield for islet preparation and transplantation in the treatment of type 1 diabetes.

ALK5 inhibition maintains islet endothelial cell survival but does not enhance islet graft revascularisation or function.

PubMed

King, A J F; Clarkin, C E; Austin, A L F; Ajram, L; Dhunna, J K; Jamil, M O; Ditta, S I; Ibrahim, S; Raza, Z; Jones, P M

2015-01-01

Islet transplantation is a potential treatment for Type 1 diabetes but long term graft function is suboptimal. The rich supply of intraislet endothelial cells diminishes rapidly after islet isolation and culture, which affects the revascularisation rate of islets after transplantation. The ALK5 pathway inhibits endothelial cell proliferation and thus inhibiting ALK5 is a potential target for improving endothelial cell survival. The aim of the study was to establish whether ALK5 inhibition prevents the loss of intraislet endothelial cells during islet culture and thus improves the functional survival of transplanted islets by enhancing their subsequent revascularisation after implantation. Islets were cultured for 48 h in the absence or presence of 2 different ALK inhibitors: SB-431542 or A-83-01. Their vascular density after culture was analysed using immunohistochemistry. Islets pre-cultured with the ALK5 inhibitors were implanted into streptozotocin-diabetic mice for either 3 or 7 days and blood glucose concentrations were monitored and vascular densities of the grafts were analysed. Islets cultured with ALK5 inhibitors had higher vascular densities than control-cultured islets. Three days after implantation, endothelial cell numbers in islet grafts were minimal, irrespective of treatment during culture. Seven days after implantation, endothelial cells were evident within the islet grafts but there was no difference between control-cultured islets and islets pre-treated with an ALK5 inhibitor. Blood glucose concentrations were no different between the treatment groups. In conclusion, inhibition of ALK5 improved intraislet endothelial cell numbers after islet culture, but this effect was lost in the early post-transplantation period. © Georg Thieme Verlag KG Stuttgart · New York.

Facilitated Engraftment of Isolated Islets Coated With Expanded Vascular Endothelial Cells for Islet Transplantation.

PubMed

Barba-Gutierrez, D Alonso; Daneri-Navarro, A; Villagomez-Mendez, J Jesus Alejandro; Kanamune, J; Robles-Murillo, A Karina; Sanchez-Enriquez, S; Villafan-Bernal, J Rafael; Rivas-Carrillo, J D

2016-03-01

Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the β-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation. We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control. Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation. We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements. Copyright © 2016 Elsevier Inc. All rights reserved.

Role of Natural Killer Cells in the Innate Immune System After Intraportal Islet Transplantation in Mice.

PubMed

Saeki, Y; Ishiyama, K; Ishida, N; Tanaka, Y; Ohdan, H

Both liver natural killer (NK) and NK T cells of the innate immune system play a crucial role in islet graft loss after intraportal islet transplantation, although a relationship between NK and NK T cells in islet loss has not been proven. In this study, we investigated the role of NK cells in the innate immune system in islet graft loss after intraportal islet transplantation. To investigate the involvement of liver NK cells in islet destruction, we assessed the differences in graft survival after intraportal islet transplantation between CD1d -/- diabetic mice and NK cell-depleted CD1d -/- diabetic mice. The transplantation of 400 islets into the liver was sufficient to reverse hyperglycemia in wild-type diabetic mice (100%, 4/4). However, normoglycemia could not be achieved when 200 islets were transplanted (0%, 0/4). In contrast, intraportal transplantation of 200 islets in NK cell-depleted CD1d -/- diabetic mice ameliorated hyperglycemia in 71% of cases (5/7), whereas transplantation of the same number of islets in CD1d -/- diabetic mice did not (0%, 0/4). Histologic findings also confirmed that intact islets were observed in NK cell-depleted CD1d -/- diabetic mice, but were difficult to observe in CD1d -/- diabetic mice. The involvement of liver NK cells in the innate immune system related to islet graft loss after intraportal islet transplantation is revealed by improved graft survival and function in NK cell-depleted CD1d -/- diabetic mice. Our data reveal that regulation of NK cell activity is particularly important when insufficient islet numbers are used for transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

LH-21 and abnormal cannabidiol improve β-cell function in isolated human and mouse islets through GPR55-dependent and -independent signalling.

PubMed

Ruz-Maldonado, Inmaculada; Pingitore, Attilio; Liu, Bo; Atanes, Patricio; Huang, Guo Cai; Baker, David; Alonso, Francisco José; Bermúdez-Silva, Francisco Javier; Persaud, Shanta J

2018-04-01

To examine the effects of Abn-CBD (GPR55 agonist) and LH-21 (CB1 antagonist) on human and mouse islet function, and to determine signalling via GPR55 using islets from GPR55 -/- mice. Islets isolated from human organ donors and mice were incubated in the absence or presence of Abn-CBD or LH-21, and insulin secretion, [Ca 2+ ] i, cAMP , apoptosis, β-cell proliferation and CREB and AKT phosphorylation were examined using standard techniques. Abn-CBD potentiated glucose-stimulated insulin secretion and elevated [Ca 2+ ] i in human islets and islets from both GPR55 +/+ and GPR55 -/- mice. LH-21 also increased insulin secretion and [Ca 2+ ] i in human islets and GPR55 +/+ mouse islets, but concentrations of LH-21 up to 0.1 μM were ineffective in islets from GPR55 -/- mice. Neither ligand affected basal insulin secretion or islet cAMP levels. Abn-CBD and LH-21 reduced cytokine-induced apoptosis in human islets and GPR55 +/+ mouse islets, and these effects were suppressed after GPR55 deletion. They also increased β-cell proliferation: the effects of Abn-CBD were preserved in islets from GPR55 -/- mice, while those of LH-21 were abolished. Abn-CBD and LH-21 increased AKT phosphorylation in mouse and human islets. This study showed that Abn-CBD and LH-21 improve human and mouse islet β-cell function and viability. Use of islets from GPR55 -/- mice suggests that designation of Abn-CBD and LH-21 as a GPR55 agonist and a CB1 antagonist, should be revised. © 2017 John Wiley & Sons Ltd.

Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability.

PubMed

Weegman, Bradley P; Kumar Sajja, Venkata Sunil; Suszynski, Thomas M; Rizzari, Michael D; Scott Iii, William E; Kitzmann, Jennifer P; Mueller, Kate R; Hanley, Thomas R; Kennedy, David J; Todd, Paul W; Balamurugan, Appakalai N; Hering, Bernhard J; Papas, Klearchos K

2016-01-01

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation.

Continuous Quadrupole Magnetic Separation of Islets during Digestion Improves Purified Porcine Islet Viability

PubMed Central

Kumar Sajja, Venkata Sunil; Rizzari, Michael D.; Scott III, William E.; Kitzmann, Jennifer P.; Kennedy, David J.; Todd, Paul W.; Balamurugan, Appakalai N.; Hering, Bernhard J.

2016-01-01

Islet transplantation (ITx) is an emerging and promising therapy for patients with uncontrolled type 1 diabetes. The islet isolation and purification processes require exposure to extended cold ischemia, warm-enzymatic digestion, mechanical agitation, and use of damaging chemicals for density gradient separation (DG), all of which reduce viable islet yield. In this paper, we describe initial proof-of-concept studies exploring quadrupole magnetic separation (QMS) of islets as an alternative to DG to reduce exposure to these harsh conditions. Three porcine pancreata were split into two parts, the splenic lobe (SPL) and the combined connecting/duodenal lobes (CDL), for paired digestions and purifications. Islets in the SPL were preferentially labeled using magnetic microparticles (MMPs) that lodge within the islet microvasculature when infused into the pancreas and were continuously separated from the exocrine tissue by QMS during the collection phase of the digestion process. Unlabeled islets from the CDL were purified by conventional DG. Islets purified by QMS exhibited significantly improved viability (measured by oxygen consumption rate per DNA, p < 0.03) and better morphology relative to control islets. Islet purification by QMS can reduce the detrimental effects of prolonged exposure to toxic enzymes and density gradient solutions and substantially improve islet viability after isolation. PMID:27843954

International workshop: islet transplantation without borders enabling islet transplantation in Greece with international collaboration and innovative technology.

PubMed

Papas, Klearchos K; Karatzas, Theodore; Berney, Thierry; Minor, Thomas; Pappas, Paris; Pattou, François; Shaw, James; Toso, Christian; Schuurman, Henk-Jan

2013-01-01

Recently, initiatives have been undertaken to establish an islet transplantation program in Athens, Greece. A major hurdle is the high cost associated with the establishment and maintenance of a clinical-grade islet manufacturing center. A collaboration was established with the University Hospitals of Geneva, Switzerland, to enable remote islet cell manufacturing with an established and validated fully operational team. However, remote islet manufacturing requires shipment of the pancreas from the procurement to the islet manufacturing site (in this case from anywhere in Greece to Geneva) and then shipment of the islets from the manufacturing site to the transplant site (from Geneva to Athens). To address challenges related to cold ischemia time of the pancreas and shipment time of islets, a collaboration was initiated with the University of Arizona, Tucson, USA. An international workshop was held in Athens, December 2011, to mark the start of this collaborative project. Experts in the field presented in three main sessions: (i) islet transplantation: state-of-the-art and the "network approach"; (ii) technical aspects of clinical islet transplantation and outcomes; and (iii) islet manufacturing - from the donated pancreas to the islet product. This manuscript presents a summary of the workshop. © 2013 John Wiley & Sons A/S.

The role of endothelial cells on islet function and revascularization after islet transplantation.

PubMed

Del Toro-Arreola, Alicia; Robles-Murillo, Ana Karina; Daneri-Navarro, Adrian; Rivas-Carrillo, Jorge David

2016-01-02

Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation.

Intra-islet endothelial cell and β-cell crosstalk: Implication for islet cell transplantation

PubMed Central

Narayanan, Siddharth; Loganathan, Gopalakrishnan; Dhanasekaran, Maheswaran; Tucker, William; Patel, Ankit; Subhashree, Venugopal; Mokshagundam, SriPrakash; Hughes, Michael G; Williams, Stuart K; Balamurugan, Appakalai N

2017-01-01

The intra-islet microvasculature is a critical interface between the blood and islet endocrine cells governing a number of cellular and pathophysiological processes associated with the pancreatic tissue. A growing body of evidence indicates a strong functional and physical interdependency of β-cells with endothelial cells (ECs), the building blocks of islet microvasculature. Intra-islet ECs, actively regulate vascular permeability and appear to play a role in fine-tuning blood glucose sensing and regulation. These cells also tend to behave as “guardians”, controlling the expression and movement of a number of important immune mediators, thereby strongly contributing to the physiology of islets. This review will focus on the molecular signalling and crosstalk between the intra-islet ECs and β-cells and how their relationship can be a potential target for intervention strategies in islet pathology and islet transplantation. PMID:28507914

A 3D map of the islet routes throughout the healthy human pancreas

PubMed Central

Ionescu-Tirgoviste, Constantin; Gagniuc, Paul A.; Gubceac, Elvira; Mardare, Liliana; Popescu, Irinel; Dima, Simona; Militaru, Manuella

2015-01-01

Islets of Langerhans are fundamental in understanding diabetes. A healthy human pancreas from a donor has been used to asses various islet parameters and their three-dimensional distribution. Here we show that islets are spread gradually from the head up to the tail section of the pancreas in the form of contracted or dilated islet routes. We also report a particular anatomical structure, namely the cluster of islets. Our observations revealed a total of 11 islet clusters which comprise of small islets that surround large blood vessels. Additional observations in the peripancreatic adipose tissue have shown lymphoid-like nodes and blood vessels captured in a local inflammatory process. Our observations are based on regional slice maps of the pancreas, comprising of 5,423 islets. We also devised an index of sphericity which briefly indicates various islet shapes that are dominant throughout the pancreas. PMID:26417671

Pancreatic islet blood flow and its measurement

PubMed Central

Jansson, Leif; Barbu, Andreea; Bodin, Birgitta; Drott, Carl Johan; Espes, Daniel; Gao, Xiang; Grapensparr, Liza; Källskog, Örjan; Lau, Joey; Liljebäck, Hanna; Palm, Fredrik; Quach, My; Sandberg, Monica; Strömberg, Victoria; Ullsten, Sara; Carlsson, Per-Ola

2016-01-01

Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting β-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future. PMID:27124642

Selective Osmotic Shock (SOS)-Based Islet Isolation for Microencapsulation.

PubMed

Enck, Kevin; McQuilling, John Patrick; Orlando, Giuseppe; Tamburrini, Riccardo; Sivanandane, Sittadjody; Opara, Emmanuel C

2017-01-01

Islet transplantation (IT) has recently been shown to be a promising alternative to pancreas transplantation for reversing diabetes. IT requires the isolation of the islets from the pancreas, and these islets can be used to fabricate a bio-artificial pancreas. Enzymatic digestion is the current gold standard procedure for islet isolation but has lingering concerns. One such concern is that it has been shown to damage the islets due to nonselective tissue digestion. This chapter provides a detailed description of a nonenzymatic method that we are exploring in our lab as an alternative to current enzymatic digestion procedures for islet isolation from human and nonhuman pancreatic tissues. This method is based on selective destruction and protection of specific cell types and has been shown to leave the extracellular matrix (ECM) of islets intact, which may thus enhance islet viability and functionality. We also show that these SOS-isolated islets can be microencapsulated for transplantation.

Islet organogenesis, angiogenesis and innervation.

PubMed

Cerf, Marlon E

2011-11-01

The pancreas is characterized by a major component, an exocrine and ductal system involved in digestion, and a minor component, the endocrine islets represented by islet micro-organs that tightly regulate glucose homoeostasis. Pancreatic organogenesis is strictly co-ordinated by transcription factors that are expressed sequentially to yield functional islets capable of maintaining glucose homoeostasis. Angiogenesis and innervation complete islet development, equipping islets to respond to metabolic demands. Proper regulation of this triad of processes during development is critical for establishing functional islets.

A peptide-major histocompatibility complex II chimera favors survival of pancreatic beta-islets grafted in type 1 diabetic mice.

PubMed

Casares, Sofia; Lin, Marvin; Zhang, Nan; Teijaro, John R; Stoica, Cristina; McEvoy, Robert; Farber, Donna L; Bona, Constantin; Brumeanu, Teodor D

2008-06-27

Transplantation of pancreatic islets showed a tremendous progress over the years as a promising, new therapeutic strategy in patients with type 1 diabetes. However, additional immunosuppressive drug therapy is required to prevent rejection of engrafted islets. The current immunosuppressive therapies showed limited success in maintaining long-term islet survival as required to achieve insulin independence in type 1 diabetes, and they induce severe adverse effects. Herein, we analyzed the effects of a soluble peptide-major histocompatibility complex (MHC) class II chimera aimed at devising an antigen-specific therapy for suppression of anti-islet T cell responses and to improve the survival of pancreatic islets transplants. Pancreatic islets from transgenic mice expressing the hemagglutinin antigen in the beta islets under the rat insulin promoter (RIP-HA) were grafted under the kidney capsule of diabetic, double transgenic mice expressing hemagglutinin in the pancreas and T cells specific for hemagglutinin (RIP-HA, TCR-HA). The recipient double transgenic mice were treated or not with the soluble peptide-MHC II chimera, and the progression of diabetes, graft survival, and T cell responses to the grafted islets were analyzed. The peptide-MHC II chimera protected syngeneic pancreatic islet transplants against the islet-reactive CD4 T cells, and prolonged the survival of transplanted islets. Protection of transplanted islets occurred by polarization of antigen-specific memory CD4 T cells toward a Th2 anti-inflammatory response. The peptide-MHC II chimera approach is an efficient and specific therapeutic approach to suppress anti-islet T cell responses and provides a long survival of pancreatic grafted islets.

Viability and functional assessment of murine pancreatic islets after transportation between Korea and Japan.

PubMed

Lee, S; Takahashi, Y; Lee, K M; Mizuno, M; Nemeno, J G; Takebe, T; Lee, J I

2015-04-01

Organ donor scarcity remains a restricting factor for pancreatic islet transplantation. To date, limited information is available on the impact of long-distance transportation on transplantable pancreatic islets. The objective of this study was to assess the effects of transportation on the viability and function of murine pancreatic islet cells. The isolated murine pancreatic islets were transported from Japan to Korea with the use of commercial modes of transportation: subway and commercial airplane. After transportation, the islets were assessed by performing a viability assay and by evaluating the islets' insulin secretion in response to glucose stimulation. A comparative study was performed for evaluating the insulin secretory responses of transported and control islets (not transported). There was no evidence of contamination in the transported pancreatic islets. No significant differences were observed in the viability and functionality of the transported and control islet cells. These findings show the feasibility of pancreatic islet transportation from Japan to Korea. Our data could be used not only for the inter-Asian but also for global advancement of animal and human islet transportation methods and transplantation research. Copyright © 2015 Elsevier Inc. All rights reserved.

Islets of Langerhans in the parakeet, Psittacula krameri.

PubMed

Gupta, Y K; Kumar, S

1980-01-01

The pancreatic gland of Psittacula krameri is divisible into 4 lobes i.e. dorsal, ventral, third and splenic. The endocrine part is composed of alpha 1-, alpha 2- and beta-cells. The islets are of 4 kinds viz., alpha islets (having alpha 1- and alpha 2-cells), beta islets (having beta- and alpha 1-cells), pure beta islets (consisting of beta-cells exclusively) and mixed islets (with beta-, alpha 1- and alpha 2-cells). The distribution of alpha islets is mostly restricted to the splenic and third lobes whereas the beta islets are found in all 4 lobes. Though the alpha islets are only few in the dorsal lobe, their size is best developed in the third and dorsal lobes. Sometimes beta and alpha islets are present in very close proximity but their cells never mingle. An interesting feature was the complete absence of alpha islets from the ventral lobe.A relative abundance of alpha 2- cells in this bird seems to be associated with its comparatively higher blood glucose level and frugivorous habit. Tinctorial reactions suggest that the insulin content of the endocrine pancreas is low. There were no seasonal changes in the islet tissue of P. krameri.

Localized internal radiotherapy with 90Y particles embedded in a new thermosetting alginate gel: a feasibility study in pigs.

PubMed

Holte, Oyvind; Skretting, Arne; Bach-Gansmo, Tore; Hol, Per Kristian; Johnsrud, Kjersti; Tønnesen, Hanne Hjorth; Karlsen, Jan

2006-02-01

Internal radiotherapy requires the localization of the radionuclide to the site of action. A new injectable alginate gel formulation intended to undergo immediate gelation in tissues and capable of encapsulating radioactive particles containing 90Y was investigated. The formulation was injected intramuscularly, into the bone marrow compartment of the femur and intravenously, respectively, in pigs. The distribution of radioactivity in various tissues was determined. Following intramuscular injection, more than 90% of the radioactivity was found at the site of injection. Following injection into bone marrow, 30-40% of the radioactivity was retained at the site of injection, but a considerable amount of radioactivity was also detected in the lungs (35-45%) and the liver (5-18%). Following intravenous injection, 80-90% of the radioactivity was found in the lungs. The present formulation appears suitable for localized radiotherapy in organs and tissues having low perfusion.

Ferroptosis-inducing agents compromise in vitro human islet viability and function.

PubMed

Bruni, Antonio; Pepper, Andrew R; Pawlick, Rena L; Gala-Lopez, Boris; Gamble, Anissa F; Kin, Tatsuya; Seeberger, Karen; Korbutt, Gregory S; Bornstein, Stefan R; Linkermann, Andreas; Shapiro, A M James

2018-05-22

Human islet transplantation has been hampered by donor cell death associated with the islet preparation procedure before transplantation. Regulated necrosis pathways are biochemically and morphologically distinct from apoptosis. Recently, ferroptosis was identified as a non-apoptotic form of iron-dependent regulated necrosis implicated in various pathological conditions. Mediators of islet oxidative stress, including glutathione peroxidase-4 (GPX4), have been identified as inhibitors of ferroptosis, and mechanisms that affect GPX4 function can impact islet function and viability. Ferroptosis has not been investigated directly in human islets, and its relevance in islet transplantation remains unknown. Herein, we sought to determine whether in vitro human islet viability and function is compromised in the presence of two distinct ferroptosis-inducing agents (FIA), erastin or RSL3, and whether these effects could be rescued with ferroptosis inhibitors, ferrostatin-1 (Fer-1), or desferrioxamine (DFO). Viability, as assessed by lactate dehydrogenase (LDH) release, revealed significant death in erastin- and RSL3-treated islets, 20.3% ± 3.8 and 24.4% ± 2.5, 24 h post culture, respectively. These effects were ameliorated in islets pre-treated with Fer-1 or the iron chelator, desferrioxamine (DFO). Stimulation index, a marker of islet function revealed a significant reduction in function in erastin-treated islets (control 1.97 ± 0.13 vs. 50 μM erastin 1.32 ± 0.1) (p < 0.05). Fer-1 and DFO pre-treatment alone did not augment islet viability or function. Pre-treatment of islets with erastin or Fer-1 did not impact in vivo engraftment in an immunodeficient mouse transplant model. Our data reveal that islets are indeed susceptible to ferroptosis in vitro, and induction of this novel cell death modality leads to compromised islet function, which can be recoverable in the presence of the ferroptosis inhibitors. The in vivo impact of this pathway in islet transplantation remains elusive given the constraints of our study, but warrants continued investigation.

Commercially Available Gas-Permeable Cell Culture Bags May Not Prevent Anoxia in Cultured or Shipped Islets

PubMed Central

Avgoustiniatos, E.S.; Hering, B.J.; Rozak, P.R.; Wilson, J.R.; Tempelman, L.A.; Balamurugan, A.N.; Welch, D.P.; Weegman, B.P.; Suszynski, T.M.; Papas, K.K.

2009-01-01

Prolonged anoxia has deleterious effects on islets. Gas-permeable cell culture devices can be used to minimize anoxia during islet culture and especially during shipment when elimination of gas-liquid interfaces is required to prevent the formation of damaging gas bubbles. Gas-permeable bags may have several drawbacks, such as propensity for puncture and contamination, difficult islet retrieval, and significantly lower oxygen permeability than silicone rubber membranes (SRM). We hypothesized that oxygen permeability of bags may be insufficient for islet oxygenation. We measured oxygen transmission rates through the membrane walls of three different types of commercially available bags and through SRM currently used for islet shipment. We found that the bag membranes have oxygen transmission rates per unit area about 100-fold lower than SRM. We solved the oxygen diffusion-reaction equation for 150-μm diameter islets seeded at 3000 islet equivalents per cm2, a density adequate to culture and ship an entire human or porcine islet preparation in a single gas-permeable device, predicting that about 40% of the islet volume would be anoxic at 22°C and about 70% would be anoxic at 37°C. Islets of larger size or islets accumulated during shipment would be even more anoxic. The model predicted no anoxia in islets similarly seeded in devices with SRM bottoms. We concluded that commercially available bags may not prevent anoxia during islet culture or shipment; devices with SRM bottoms are more suitable alternatives. PMID:18374080

Preimplantation of an immunoprotective device can lower the curative dose of islets to that of free islet transplantation: studies in a rodent model.

PubMed

Sörenby, Anne K; Kumagai-Braesch, Makiko; Sharma, Amit; Hultenby, Kjell R; Wernerson, Annika M; Tibell, Annika B

2008-07-27

Islet graft survival inside macroencapsulation devices is suboptimal. We hypothesized that induction of neovascularization by preimplantation of devices would improve the physiological conditions, thereby lowering the number of islets required for cure. Several rat islets were transplanted to TheraCyte immunoprotective devices implanted subcutaneously in diabetic athymic mice. Cure rates in the groups with preimplanted devices were significantly better than in those with freshly implanted devices (375 islets: 8/8 vs. 1/6, P=0.003; 125 islets: 6/6 vs. 0/7, P=0.001). Morphometric evaluations of the 125 islet groups showed higher fractional and absolute volumes of endocrine tissue in the group with preimplanted devices (P<0.001 and P=0.035, respectively). In the following dose titration study, using preimplanted devices, as low as 50 islets cured diabetic mice (100% cure, n=6). We conclude that preimplantation significantly lowers the curative dose of macroencapsulated islets to levels resembling those of free islets transplanted under the renal capsule.

Amyloid formation reduces protein kinase B phosphorylation in primary islet β-cells which is improved by blocking IL-1β signaling

PubMed Central

Zhang, Yun; Warnock, Garth L.; Ao, Ziliang; Park, Yoo Jin; Safikhan, Nooshin; Ghahary, Aziz

2018-01-01

Amyloid formation in the pancreatic islets due to aggregation of human islet amyloid polypeptide (hIAPP) contributes to reduced β-cell mass and function in type 2 diabetes (T2D) and islet transplantation. Protein kinase B (PKB) signaling plays a key role in the regulation of β-cell survival, function and proliferation. In this study, we used human and hIAPP-expressing transgenic mouse islets in culture as two ex vivo models of human islet amyloid formation to: 1. Investigate the effects of amyloid formation on PKB phosphorylation in primary islet β-cells; 2. Test if inhibition of amyloid formation and/or interleukin-1β (IL-1β) signaling in islets can restore the changes in β-cell phospho-PKB levels mediated by amyloid formation. Human and hIAPP-expressing mouse islets were cultured in elevated glucose with an amyloid inhibitor (Congo red) or embedded within collagen matrix to prevent amyloid formation. To block the IL-1β signaling, human islets were treated with an IL-1 receptor antagonist (anakinra) or a glucagon-like peptide-1 agonist (exenatide). β-cell phospho-PKB levels, proliferation, apoptosis, islet IL-1β levels and amyloid formation were assessed. Amyloid formation in both cultured human and hIAPP-expressing mouse islets reduced β-cell phospho-PKB levels and increased islet IL-1β levels, both of which were restored by prevention of amyloid formation either by the amyloid inhibitor or embedding islets in collagen matrix, resulting in improved β-cell survival. Furthermore, inhibition of IL-1β signaling by treatment with anakinra or exenatide increased β-cell phospho-PKB levels, enhanced proliferation and reduced apoptosis in amyloid forming human islets during 7-day culture. These data suggest that amyloid formation leads to reduced PKB phosphorylation in β-cells which is associated with elevated islet IL-1β levels. Inhibitors of amyloid or amyloid-induced IL-1β production may provide a new approach to restore phospho-PKB levels thereby enhance β-cell survival and proliferation in conditions associated with islet amyloid formation such as T2D and clinical islet transplantation. PMID:29474443

Extracellular Matrix and Growth Factors Improve the Efficacy of Intramuscular Islet Transplantation.

PubMed

Tsuchiya, Haruyuki; Sakata, Naoaki; Yoshimatsu, Gumpei; Fukase, Masahiko; Aoki, Takeshi; Ishida, Masaharu; Katayose, Yu; Egawa, Shinichi; Unno, Michiaki

2015-01-01

The efficacy of intramuscular islet transplantation is poor despite being technically simple, safe, and associated with reduced rates of severe complications. We evaluated the efficacy of combined treatment with extracellular matrix (ECM) and growth factors in intramuscular islet transplantation. Male BALB/C mice were used for the in vitro and transplantation studies. The following three groups were evaluated: islets without treatment (islets-only group), islets embedded in ECM with growth factors (Matrigel group), and islets embedded in ECM without growth factors [growth factor-reduced (GFR) Matrigel group]. The viability and insulin-releasing function of islets cultured for 96 h were significantly improved in Matrigel and GFR Matrigel groups compared with the islets-only group. Blood glucose and serum insulin levels immediately following transplantation were significantly improved in the Matrigel and GFR Matrigel groups and remained significantly improved in the Matrigel group at postoperative day (POD) 28. On histological examination, significantly decreased numbers of TdT-mediated deoxyuridine triphosphate-biotin nick end labeling-positive islet cells and significantly increased numbers of Ki67-positive cells were observed in the Matrigel and GFR Matrigel groups at POD 3. Peri-islet revascularization was most prominent in the Matrigel group at POD 14. The efficacy of intramuscular islet transplantation was improved by combination treatment with ECM and growth factors through the inhibition of apoptosis, increased proliferation of islet cells, and promotion of revascularization.

Glucose diffusion in pancreatic islets of Langerhans.

PubMed Central

Bertram, R; Pernarowski, M

1998-01-01

We investigate the time required for glucose to diffuse through an isolated pancreatic islet of Langerhans and reach an equilibrium. This question is relevant in the context of in vitro electrophysiological studies of the response of an islet to step changes in the bath glucose concentration. Islet cells are electrically coupled by gap junctions, so nonuniformities in islet glucose concentration may be reflected in the activity of cells on the islet periphery, where electrical recordings are made. Using a mathematical model of hindered glucose diffusion, we investigate the effects of the islet porosity and the permeability of a surrounding layer of acinar cells. A major factor in the determination of the equilibrium time is the transport of glucose into islet beta-cells, which removes glucose from the interstitial spaces where diffusion occurs. This transport is incorporated by using a model of the GLUT-2 glucose transporter. We find that several minutes are required for the islet to equilibrate to a 10 mM change in bath glucose, a typical protocol in islet experiments. It is therefore likely that in electrophysiological islet experiments the glucose distribution is nonuniform for several minutes after a step change in bath glucose. The delay in glucose penetration to the inner portions of the islet may be a major contributing factor to the 1-2-min delay in islet electrical activity typically observed after bath application of a stimulatory concentration of glucose. PMID:9545035

Effect of the Diabetic State on Islet Engraftment and Function in a Large Animal Model of Islet-Kidney Transplantation.

PubMed

Vallabhajosyula, Prashanth; Hirakata, Atsushi; Weiss, Matthew; Griesemer, Adam; Shimizu, Akira; Hong, Hanzhou; Habertheuer, Andreas; Tchipashvili, Vaja; Yamada, Kazuhiko; Sachs, David H

2017-11-01

In islet transplantation, in addition to immunologic and ischemic factors, the diabetic/hyperglycemic state of the recipient has been proposed, although not yet validated, as a possible cause of islet toxicity, contributing to islet loss during the engraftment period. Using a miniature swine model of islet transplantation, we have now assessed the effect of a persistent state of hyperglycemia on islet engraftment and subsequent function. An islet-kidney (IK) model previously described by our laboratory was utilized. Three experimental donor animals underwent total pancreatectomy and autologous islet transplantation underneath the renal capsule to prepare an IK at a load of ≤1,000 islet equivalents (IE)/kg donor weight, leading to a chronic diabetic state during the engraftment period (fasting blood glucose >250 mg/dL). Three control donor animals underwent partial pancreatectomy (sufficient to maintain normoglycemia during islet engraftment period) and IK preparation. As in vivo functional readout for islet engraftment, the IKs were transplanted across an immunologic minor or class I mismatch barrier into diabetic, nephrectomized recipients at an islet load of ∼4,500 IE/kg recipient weight. A 12-d course of cyclosporine was administered for tolerance induction. All experimental donors became diabetic and showed signs of end organ injury, while control donors maintained normoglycemia. All recipients of IK from both experimental and control donors achieved glycemic control over long-term follow-up, with reversal of diabetic nephropathy and with similar glucose tolerance tests. In this preclinical, large animal model, neither islet engraftment nor subsequent long-term islet function after transplantation appear to be affected by the diabetic state.

Insulin-Like growth factor-II (IGF-II) prevents proinflammatory cytokine-induced apoptosis and significantly improves islet survival after transplantation.

PubMed

Hughes, Amy; Mohanasundaram, Daisy; Kireta, Svjetlana; Jessup, Claire F; Drogemuller, Chris J; Coates, P Toby H

2013-03-15

The early loss of functional islet mass (50-70%) due to apoptosis after clinical transplantation contributes to islet allograft failure. Insulin-like growth factor (IGF)-II is an antiapoptotic protein that is highly expressed in β-cells during development but rapidly decreases in postnatal life. We used an adenoviral (Ad) vector to overexpress IGF-II in isolated rat islets and investigated its antiapoptotic action against exogenous cytokines interleukin-1β- and interferon-γ-induced islet cell death in vitro. Using an immunocompromised marginal mass islet transplant model, the ability of Ad-IGF-II-transduced rat islets to restore euglycemia in nonobese diabetic/severe combined immunodeficient diabetic recipients was assessed. Ad-IGF-II transduction did not affect islet viability or function. Ad-IGF-II cytokine-treated islets exhibited decreased cell death (40% ± 2.8%) versus Ad-GFP and untransduced control islets (63.2% ± 2.5% and 53.6% ± 2.3%, respectively). Ad-IGF-II overexpression during cytokine treatment resulted in a marked reduction in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive apoptotic cells (8.3% ± 1.4%) versus Ad-GFP control (41% ± 4.2%) and untransduced control islets (46.5% ± 6.2%). Western blot analysis confirmed that IGF-II inhibits apoptosis via activation of the phosphatidylinositol 3-kinase/Akt signaling pathway. Transplantation of IGF-II overexpressing islets under the kidney capsule of diabetic mice restored euglycemia in 77.8% of recipients compared with 18.2% and 47.5% of Ad-GFP and untransduced control islet recipients, respectively (P<0.05, log-rank [Mantel-Cox] test). Antiapoptotic IGF-II decreases apoptosis in vitro and significantly improved islet transplant outcomes in vivo. Antiapoptotic gene transfer is a potentially powerful tool to improve islet survival after transplantation.

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.)

PubMed Central

He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

2015-01-01

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution. PMID:25682842

Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

PubMed

He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

2015-04-01

Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution. © 2015 Anatomical Society.

Comparison of volume estimation methods for pancreatic islet cells

NASA Astrophysics Data System (ADS)

Dvořák, JiřÃ.­; Å vihlík, Jan; Habart, David; Kybic, Jan

2016-03-01

In this contribution we study different methods of automatic volume estimation for pancreatic islets which can be used in the quality control step prior to the islet transplantation. The total islet volume is an important criterion in the quality control. Also, the individual islet volume distribution is interesting -- it has been indicated that smaller islets can be more effective. A 2D image of a microscopy slice containing the islets is acquired. The input of the volume estimation methods are segmented images of individual islets. The segmentation step is not discussed here. We consider simple methods of volume estimation assuming that the islets have spherical or ellipsoidal shape. We also consider a local stereological method, namely the nucleator. The nucleator does not rely on any shape assumptions and provides unbiased estimates if isotropic sections through the islets are observed. We present a simulation study comparing the performance of the volume estimation methods in different scenarios and an experimental study comparing the methods on a real dataset.

Oxygen-permeable microwell device maintains islet mass and integrity during shipping

PubMed Central

Rojas-Canales, Darling M; Waibel, Michaela; Forget, Aurelien; Penko, Daniella; Nitschke, Jodie; Harding, Fran J; Delalat, Bahman; Blencowe, Anton; Loudovaris, Thomas; Grey, Shane T; Thomas, Helen E; Kay, Thomas W H; Drogemuller, Chris J; Voelcker, Nicolas H; Coates, Patrick T

2018-01-01

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20–40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping. PMID:29483160

Oxygen-permeable microwell device maintains islet mass and integrity during shipping.

PubMed

Rojas-Canales, Darling M; Waibel, Michaela; Forget, Aurelien; Penko, Daniella; Nitschke, Jodie; Harding, Fran J; Delalat, Bahman; Blencowe, Anton; Loudovaris, Thomas; Grey, Shane T; Thomas, Helen E; Kay, Thomas W H; Drogemuller, Chris J; Voelcker, Nicolas H; Coates, Patrick T

2018-03-01

Islet transplantation is currently the only minimally invasive therapy available for patients with type 1 diabetes that can lead to insulin independence; however, it is limited to only a small number of patients. Although clinical procedures have improved in the isolation and culture of islets, a large number of islets are still lost in the pre-transplant period, limiting the success of this treatment. Moreover, current practice includes islets being prepared at specialized centers, which are sometimes remote to the transplant location. Thus, a critical point of intervention to maintain the quality and quantity of isolated islets is during transportation between isolation centers and the transplanting hospitals, during which 20-40% of functional islets can be lost. The current study investigated the use of an oxygen-permeable PDMS microwell device for long-distance transportation of isolated islets. We demonstrate that the microwell device protected islets from aggregation during transport, maintaining viability and average islet size during shipping. © 2018 The authors.

Pancreatic islet enhancer clusters enriched in type 2 diabetes risk-associated variants.

PubMed

Pasquali, Lorenzo; Gaulton, Kyle J; Rodríguez-Seguí, Santiago A; Mularoni, Loris; Miguel-Escalada, Irene; Akerman, İldem; Tena, Juan J; Morán, Ignasi; Gómez-Marín, Carlos; van de Bunt, Martijn; Ponsa-Cobas, Joan; Castro, Natalia; Nammo, Takao; Cebola, Inês; García-Hurtado, Javier; Maestro, Miguel Angel; Pattou, François; Piemonti, Lorenzo; Berney, Thierry; Gloyn, Anna L; Ravassard, Philippe; Skarmeta, José Luis Gómez; Müller, Ferenc; McCarthy, Mark I; Ferrer, Jorge

2014-02-01

Type 2 diabetes affects over 300 million people, causing severe complications and premature death, yet the underlying molecular mechanisms are largely unknown. Pancreatic islet dysfunction is central in type 2 diabetes pathogenesis, and understanding islet genome regulation could therefore provide valuable mechanistic insights. We have now mapped and examined the function of human islet cis-regulatory networks. We identify genomic sequences that are targeted by islet transcription factors to drive islet-specific gene activity and show that most such sequences reside in clusters of enhancers that form physical three-dimensional chromatin domains. We find that sequence variants associated with type 2 diabetes and fasting glycemia are enriched in these clustered islet enhancers and identify trait-associated variants that disrupt DNA binding and islet enhancer activity. Our studies illustrate how islet transcription factors interact functionally with the epigenome and provide systematic evidence that the dysregulation of islet enhancers is relevant to the mechanisms underlying type 2 diabetes.

Inactivation of p27kip1 Promoted Nonspecific Inflammation by Enhancing Macrophage Proliferation in Islet Transplantation.

PubMed

Li, Yang; Ding, Xiaoming; Fan, Ping; Guo, Jian; Tian, Xiaohui; Feng, Xinshun; Zheng, Jin; Tian, Puxun; Ding, Chenguang; Xue, Wujun

2016-11-01

Islet transplantation suffers from low efficiency caused by nonspecific inflammation-induced graft loss after transplantation. This study reports increased islet loss and enhanced inflammatory response in p27-deficient mice (p27-/-) and proposes a possible mechanism. Compared with wild type, p27-/- mice showed more severe functional injury of islet, with increased serum levels of inflammatory cytokines IL-1 and TNF-α, inducing macrophage proliferation. Furthermore, the increased number, proapoptotic proteins, and nuclear factor-kappa b (NF-κB) phosphorylation status of the infiltrating macrophages were accompanied by increased TNF-α mRNA level of islet graft site in p27-/- mice. Moreover, in vitro, we found that macrophages were still activated and cocultured with islet and promoted islet loss even blocking the direct effect of TNF-α on islets. Malondialdehyde (MDA, an end product of lipid peroxidation) in islet and media were increased after cocultured with macrophages. p27 deficiency also increased macrophage proliferation and islet injury. Therefore, p27 inactivation promotes injury islet graft loss via the elevation of proliferation and inflammatory cytokines secretion in infiltrating macrophages which induced nonspecific inflammation independent of TNF-α/nuclear factor-kappa b pathway. This potentially represents a promising therapeutic target in improving islet graft survival.

Beneficial effect of D-allose for isolated islet culture prior to islet transplantation.

PubMed

Kashiwagi, Hirotaka; Asano, Eisuke; Noguchi, Chisato; Sui, Li; Hossain, Akram; Akamoto, Shintaro; Okano, Keiichi; Tokuda, Masaaki; Suzuki, Yasuyuki

2016-01-01

Pretransplant restoration of islets damaged during isolation remains to be solved. In this study, we examined the effect of D-allose on islets isolated from rat pancreata prior to islet transplantation. Rat islets isolated from fresh pancreata were cultured overnight in Roswell Park Memorial Institute 1640 solution in the absence (group 1) or presence (group 2) of D-allose. Then we assessed stimulation index of insulin, and cure rate after islet transplantation to diabetic nude mice. We also measured malondialdehyde level and caspase 3 activity of islets after the overnight culture for assessment of the oxidative stress and the apoptosis. D-allose significantly improved insulin secretion of islets. The stimulation index in group 2 was significantly higher than in group 1. Cure rate after transplantation in group 2 was higher than in group 1 especially in the first week. The malondialdehyde level in group 2 was significantly lower than in group 1. But the caspase 3 activities in both groups did not differ. D-allose treatment of isolated islet culture prior to transplantation restored islet function and increased successful transplant rate. The results of this study suggested that D-allose improved function of damaged islets through its anti-oxidative activity. © 2015 Japanese Society of Hepato-Biliary-Pancreatic Surgery.

CCR7 directs the recruitment of T cells into inflamed pancreatic islets of nonobese diabetic (NOD) mice.

PubMed

Shan, Zhongyan; Xu, Baohui; Mikulowska-Mennis, Anna; Michie, Sara A

2014-05-01

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of insulin-producing β cells in the pancreatic islets. The migration of T cells from blood vessels into pancreas is critical for the development of islet inflammation and β cell destruction in T1D. To define the roles of C-C chemokine receptor type 7 (CCR7) in recruitment of T cells into islets, we used laser capture microdissection to isolate tissue from inflamed islets of nonobese diabetic (NOD) mice and uninflamed islets of BALB/c and young NOD mice. RT-PCR analyses detected mRNAs for CCR7 and its chemokine ligands CCL19 (ELC; MIP-3β) and CCL21 (SLC) in captures from inflamed, but not from uninflamed, islets. Immunohistology studies revealed that high endothelial venules in inflamed islets co-express CCL21 protein and MAdCAM-1 (an adhesion molecule that recruits lymphocytes into islets). Desensitization of lymphocyte CCR7 blocked about 75 % of T cell migration from the bloodstream into inflamed islets, but had no effect on B cell migration into islets. These results indicate that CCR7 and its ligands are important in the recruitment of T cells into inflamed islets and thus in the pathogenesis of T1D.

Clinical islet transplantation experience of the University of California Islet Transplant Consortium.

PubMed

Brunicardi, F C; Atiya, A; Stock, P; Kenmochi, T; Une, S; Benhamou, P Y; Watt, P C; Miyamato, M; Wantanabe, Y; Nomura, Y

1995-12-01

The University of California Islet Transplant Consortium was formed to evaluate the feasibility of performing clinical islet transplantation at different transplant centers by using a single centralized islet isolation laboratory. From July 1992 through February 1995 seven adult islet transplantations were performed, six allografts and one autograft. Once procured, human pancreata were brought to the UCLA-VA Islet Core Laboratory for islet isolation and purification, which were then transported to different centers for transplantation. Patients 1 through 3 received their transplants in Los Angeles, patient 4 received her islet transplant in Torrance, and patients 5 through 7 received their transplants in San Francisco. Although none of these patients achieved insulin independence, four of seven had functioning grafts longer than 6 months as indicated by circulating C-peptide level greater than 0.7 ng/ml. Furthermore, improved glucose control as shown by a decreased insulin requirement was seen in 57% (four of seven patients) of these patients. The ability to isolate islets at a single laboratory and transport them long distances to different centers was shown in patients 4 through 7. Islet transplantation can be performed with improvements in blood glucose control, and islets can be isolated at a centralized location and successfully transported to different centers for transplantation.

Vascularized tissue-engineered chambers promote survival and function of transplanted islets and improve glycemic control.

PubMed

Knight, K R; Uda, Y; Findlay, M W; Brown, D L; Cronin, K J; Jamieson, E; Tai, T; Keramidaris, E; Penington, A J; Rophael, J; Harrison, L C; Morrison, W A

2006-03-01

We have developed a chamber model of islet engraftment that optimizes islet survival by rapidly restoring islet-extracellular matrix relationships and vascularization. Our aim was to assess the ability of syngeneic adult islets seeded into blood vessel-containing chambers to correct streptozotocin-induced diabetes in mice. Approximately 350 syngeneic islets suspended in Matrigel extracellular matrix were inserted into chambers based on either the splenic or groin (epigastric) vascular beds, or, in the standard approach, injected under the renal capsule. Blood glucose was monitored weekly for 7 weeks, and an intraperitoneal glucose tolerance test performed at 6 weeks in the presence of the islet grafts. Relative to untreated diabetic animals, glycemic control significantly improved in all islet transplant groups, strongly correlating with islet counts in the graft (P<0.01), and with best results in the splenic chamber group. Glycemic control deteriorated after chambers were surgically removed at week 8. Immunohistochemistry revealed islets with abundant insulin content in grafts from all groups, but with significantly more islets in splenic chamber grafts than the other treatment groups (P<0.05). It is concluded that hyperglycemia in experimental type 1 diabetes can be effectively treated by islets seeded into a vascularized chamber functioning as a "pancreatic organoid."

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

PubMed

Mohammadi Ayenehdeh, Jamal; Niknam, Bahareh; Hashemi, Seyed Mahmoud; Rahavi, Hossein; Rezaei, Nima; Soleimani, Masoud; Tajik, Nader

2017-07-01

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. 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. 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. In the model presented here, the rapid islet isolation technique and the application of biomimetic hydrogel wrapping of islets could facilitate islet transplantation procedures.

Extensive Loss of Islet Mass Beyond the First Day After Intraportal Human Islet Transplantation in a Mouse Model.

PubMed

Liljebäck, Hanna; Grapensparr, Liza; Olerud, Johan; Carlsson, Per-Ola

2016-01-01

Clinical islet transplantation is characterized by a progressive deterioration of islet graft function, which renders many patients once again dependent on exogenous insulin administration within a couple of years. In this study, we aimed to investigate possible engraftment factors limiting the survival and viability of experimentally transplanted human islets beyond the first day after their transplantation to the liver. Human islets were transplanted into the liver of nude mice and characterized 1 or 30 days after transplantation by immunohistochemistry. The factors assessed were endocrine mass, cellular death, hypoxia, vascular density and amyloid formation in the transplanted islets. One day posttransplantation, necrotic cells, as well as apoptotic cells, were commonly observed. In contrast to necrotic death, apoptosis rates remained high 1 month posttransplantation, and the total islet mass was reduced by more than 50% between 1 and 30 days posttransplantation. Islet mass at 30 days posttransplantation correlated negatively to apoptotic death. Vascular density within the transplanted islets remained less than 30% of that in native human islets up to 30 days posttransplantation and was associated with prevailing hypoxia. Amyloid formation was rarely observed in the 1-day-old transplants, but was commonly observed in the 30-day-old islet transplants. We conclude that substantial islet cell death occurs beyond the immediate posttransplantation phase, particularly through apoptotic events. Concomitant low vascularization with prevailing hypoxia and progressive amyloid development was observed in the human islet grafts. Strategies to improve engraftment at the intraportal site or change of implantation site in the clinical setting are needed.

Sequential kidney/islet transplantation using prednisone-free immunosuppression.

PubMed

Kaufman, Dixon B; Baker, Marshall S; Chen, Xiaojuan; Leventhal, Joseph R; Stuart, Frank P

2002-08-01

Islet transplantation is becoming established as a treatment option for type I diabetes in select patients. Individuals with type I diabetes who have previously received a successful kidney allograft may be good candidates for islet transplantation. They have already assumed the risks of chronic immunosuppression, so the added procedural risk of a subsequent islet transplant would be minimal. Furthermore, because of the preimmunosuppressed state it is possible that islet-after-kidney transplantation may result in a more efficient early islet engraftment. Consequently, insulin independence might be achieved with significantly fewer islets than the approximately 8-10,000 islet equivalents/kg/b.w. currently required. A mass that usually demands two or more cadaveric donors. A case of successful islet-after-kidney transplantation is described using the steroid-free Edmonton immunosuppression protocol. Characteristics of the final islet product are: a) islet equivalents: 265,888 (4100 islet equivalents/kg/b.w.); b) islet purity: 75-80%; c) viability: >95% (trypan blue exclusion); and d) mean islet potency (static low-high glucose challenge): 4.16 +/- 1.91-fold increase. Post-transplant the patient's hypoglycemic episodes abated. Exogenous insulin requirements were eliminated at week 12 post-transplant as basal and Ensure (Abbott Laboratories, Abbott Park, IL, USA) oral glucose stimulated C-peptide levels peaked and stabilized. Twenty-four-hour continuous glucose monitoring confirmed moment-to-moment glycemic control, and periodic nonfasting finger stick glucose determinations over the next month confirmed glycemia was controlled. Hemoglobin A1c levels declined from a pretransplant level of 6.9% to 5.3%. Renal allograft function remained changed.

Selective Osmotic Shock for Islet Isolation in the Cadaveric Canine Pancreas.

PubMed

Thompson, Elizabeth M; Sollinger, Jennifer L; Opara, Emmanuel C; Adin, Christopher A

2018-03-01

Currently, islet isolation is performed using harsh collagenases that cause nonspecific injury to both islets and exocrine tissue, negatively affecting the outcome of cell transplantation. We evaluated a novel islet isolation protocol utilizing high concentrations of glucose to cause selective osmotic shock (SOS). Islets have a membrane glucose transporter that allows adaptation to changes in glucose concentrations while exocrine tissue can be selectively destroyed by these osmolar shifts. Canine pancreata were obtained within 15 min after euthanasia from animals ( n = 6) euthanized for reasons unrelated to this study. Each pancreas was divided into 4 segments that were randomized to receive 300 mOsm glucose for 20 min (group 1), 600 mOsm for 20 min (group 2), 300 mOsm for 40 min (group 3), or 600 mOsm for 40 min (group 4). Islet yield, purity, and viability were compared between groups. Mean ± standard error of the mean islet yield for groups 1 to 4 was 428 ± 159, 560 ± 257, 878 ± 443, and 990 ± 394 islet equivalents per gram, respectively. Purity ranged from 37% to 45% without the use of density gradient centrifugation and was not significantly different between groups. Islet cell viability was excellent overall (89%) and did not differ between treatment protocol. Islet function was best in groups treated with 300 mOsm of glucose (stimulation index [SI] = 3.3), suggesting that the lower concentration of glucose may be preferred for use in canine islet isolation. SOS provides a widely available means for researchers to isolate canine islets for use in islet transplantation or in studies of canine islet physiology.

Micro-fabricated scaffolds lead to efficient remission of diabetes in mice.

PubMed

Buitinga, Mijke; Assen, Frank; Hanegraaf, Maaike; Wieringa, Paul; Hilderink, Janneke; Moroni, Lorenzo; Truckenmüller, Roman; van Blitterswijk, Clemens; Römer, Gert-Willem; Carlotti, Françoise; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

2017-08-01

Despite the clinical success of intrahepatic islet transplantation in treating type 1 diabetes, factors specific to this transplantation site hinder long-term insulin independence. The adoption of alternative, extravascular sites likely improve islet survival and function, but few locations are able to sufficiently confine islets in order to facilitate engraftment. This work describes a porous microwell scaffold with a well-defined pore size and spacing designed to guarantee islet retention at an extrahepatic transplantation site and facilitate islet revascularization. Three techniques to introduce pores were characterized: particulate leaching; solvent casting on pillared wafers; and laser drilling. Our criteria of a maximum pore diameter of 40 μm were best achieved via laser drilling. Transplantation studies in the epididymal fat of diabetic mice elucidated the potential of this porous scaffold platform to restore blood glucose levels and facilitate islet engraftment. Six out of eight mice reverted to stable normoglycemia with a mean time to remission of 6.2 ± 3.2 days, which was comparable to that of the gold standard of renal subcapsular islet grafts. In contrast, when islets were transplanted in the epididymal fat pad without a microwell scaffold, only two out of seven mice reverted to stable normoglycemia. Detailed histological evaluation four weeks after transplantation found a comparable vascular density in scaffold-seeded islets, renal subcapsular islets and native pancreatic islets. However, the vascularization pattern in scaffold-seeded islets was more inhomogeneous compared to native pancreatic islets with a higher vascular density in the outer shell of the islets compared to the inner core. We also observed a corresponding decrease in the beta-cell density in the islet core. Despite this, our data indicated that islets transplanted in the microwell scaffold platform were able to maintain a viable beta-cell population and restore glycemic control. Furthermore, we demonstrated that the microwell scaffold platform facilitated detailed analysis at a subcellular level to correlate design parameters with functional physiological observations. Copyright © 2017 Elsevier Ltd. All rights reserved.

Mass transfer of large molecules through collagen and collagen-silica hybrid membranes

NASA Astrophysics Data System (ADS)

Jofre-Lora, Pedro

Diabetes is a growing concern in the United States and around the world that must be addressed through new treatment options. Current standard treatment options of diabetes are limiting and have tremendous impacts on patient's lives. Emerging therapies, such as the implantation of encapsulated islets, are promising treatment options, but have not yet materialized due to unsolved problems with material properties. Hybrid silica-collagen membranes address some of these unsolved problems and are a promising material for cell encapsulation. However, the mass transfer properties of large molecules, such as insulin, TNF-alpha, IL1beta, and other important proteins in the etiology of diabetes, through these hybrid membranes are poorly characterized. In order to begin characterizing these properties, a device was constructed to accurately and efficiently measure the mass transfer of other similar large molecules, fluorescein isothiocyanate dextrans (FITC-dextran), through collagen-silica hybrid membranes. The device was used to measure diffusion coefficients of 4, 20, 40, and 150 kDa FITC-dextrans through non-silicified and silicified samples of 200 and 1000 Pa porcine skin collagen. Diffusion coefficients were found to be in the 10-7-10-6 cm2s -1 range, which is in agreement with previously published data for similar molecules through similar hydrogels. The effects of collagen stiffness, FITC-dextran molecular weight, and silicification treatment on diffusion were investigated. It was found that collagen stiffness and FITC-dextran molecular weight had a negative correlation with diffusion, whereas silicification treatment had no global impact on diffusion. The device created, and the results of this preliminary investigation, can be used to develop collagen-silica hybrid membranes as an alternative material for cell encapsulation in a forward-design manner.

Mesenchymal stromal cells improve human islet function through released products and extracellular matrix.

PubMed

Arzouni, Ahmed A; Vargas-Seymour, Andreia; Rackham, Chloe L; Dhadda, Paramjeet; Huang, Guo-Cai; Choudhary, Pratik; Nardi, Nance; King, Aileen J F; Jones, Peter M

2017-12-01

The aims of the present study were (i) to determine whether the reported beneficial effects of mesenchymal stromal cells (MSCs) on mouse islet function extend to clinically relevant human tissues (islets and MSCs), enabling translation into improved protocols for clinical human islet transplantation; and (ii) to identify possible mechanisms through which human MSCs influence human islet function. Human islets were co-cultured with human adipose tissue-derived MSCs (hASCs) or pre-treated with its products - extracellular matrix (ECM) and annexin A1 (ANXA1). Mouse islets were pre-treated with mouse MSC-derived ECM. Islet insulin secretory function was assessed in vitro by radioimmunoassay. Quantitative RT-PCR was used to screen human adipMSCs for potential ligands of human islet G-protein-coupled receptors. We show that co-culture with hASCs improves human islet secretory function in vitro , as measured by glucose-stimulated insulin secretion, confirming previous reports using rodent tissues. Furthermore, we demonstrate that these beneficial effects on islet function can be partly attributed to the MSC-derived products ECM and ANXA1. Our results suggest that hASCs have the potential to improve the quality of human islets isolated for transplantation therapy of Type 1 diabetes. Furthermore, it may be possible to achieve improvements in human islet quality in a cell-free culture system by using the MSC-derived products ANXA1 and ECM. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

A brief history of clinical xenotransplantation.

PubMed

Cooper, David K C; Ekser, Burcin; Tector, A Joseph

2015-11-01

Between the 17th and 20th centuries, blood was transfused from various animal species into patients with a variety of pathological conditions. Skin grafts were carried out in the 19th century, with grafts from a variety of animals, with frogs being the most popular. In the 1920s, Voronoff advocated the transplantation of slices of chimpanzee testis into elderly men, believing that the hormones produced by the testis would rejuvenate his patients. In 1963-4, when human organs were not available and dialysis was not yet in use, Reemtsma transplanted chimpanzee kidneys into 13 patients, one of whom returned to work for almost 9 months before suddenly dying from what was believed to be an electrolyte disturbance. The first heart transplant in a human ever performed was by Hardy in 1964, using a chimpanzee heart, but the patient died within 2 h. Starzl carried out the first chimpanzee-to-human liver transplantation in 1966; in 1992 he obtained patient survival for 70 days following a baboon liver transplant. The first clinical pig islet transplant was carried out by Groth in 1993. Today, genetically-modified pigs offer hope of a limitless supply of organs and cells for those in need of a transplant. Copyright © 2015 IJS Publishing Group Limited. Published by Elsevier Ltd. All rights reserved.

Nicotinamide mononucleotide protects against pro-inflammatory cytokine-mediated impairment of mouse islet function.

PubMed

Caton, P W; Kieswich, J; Yaqoob, M M; Holness, M J; Sugden, M C

2011-12-01

Nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD(+) biosynthesis, exists as intracellular NAMPT (iNAMPT) and extracellular NAMPT (eNAMPT). eNAMPT, secreted from adipose tissue, promotes insulin secretion. Administration of nicotinamide mononucleotide (NMN), a product of the eNAMPT reaction, corrects impaired islet function in Nampt ( +/- ) mice. One of its potential targets is the NAD(+)-dependent deacetylase sirtuin 1. We hypothesised that altered NAMPT activity might contribute to the suppression of islet function associated with inflammation, and aimed to determine whether NMN could improve cytokine-mediated islet dysfunction. Acute effects of NMN on cytokine-mediated islet dysfunction were examined in islets incubated with TNFα and IL1β, and in mice fed a fructose-rich diet (FRD) for 16 weeks. Changes in iNAMPT, eNAMPT and inflammation levels were determined in FRD-fed mice. FRD-fed mice displayed markedly lower levels of circulating eNAMPT, with impaired insulin secretion and raised islet expression of Il1b. NMN administration lowered Il1b expression and restored suppressed insulin secretion in FRD-fed mice. NMN also restored insulin secretion in islets cultured with pro-inflammatory cytokines. The changes in islet function corresponded with changes in key markers of islet function and differentiation. The anti-inflammatory effects of NMN were partially blocked by inhibition of sirtuin 1. Chronic fructose feeding causes severe islet dysfunction in mice. Onset of beta cell failure in FRD-fed mice may occur via lowered secretion of eNAMPT, leading to increased islet inflammation and impaired beta cell function. Administration of exogenous NMN to FRD-fed mice corrects inflammation-induced islet dysfunction. Modulation of this pathway may be an attractive target for amelioration of islet dysfunction associated with inflammation.

Rat pancreatic islet size standardization by the "hanging drop" technique.

PubMed

Cavallari, G; Zuellig, R A; Lehmann, R; Weber, M; Moritz, W

2007-01-01

Rejection and hypoxia are the main factors that limit islet engraftment in the recipient liver in the immediate posttransplant period. Recently authors have reported a negative relationship of graft function and islet size, concluding that small islets are superior to large islets. Islets can be dissociated into single cells and reaggregated into so called "pseudoislets," which are functionally equivalent to intact islets but exhibit reduced immunogenicity. The aim of our study was develop a technique that enabled one to obtain pseudoislets of defined, preferably small, dimensions. Islets were harvested from Lewis rats by the collagenase digestion procedure. After purification, the isolated islets were dissociated into single cells by trypsin digestion. Fractions with different cell numbers were seeded into single drops onto cell culture dishes, which were inverted and incubated for 5 to 8 days under cell culture conditions. Newly formed pseudoislets were analyzed for dimension, morphology, and cellular composition. The volume of reaggregated pseudoislets strongly correlated with the cell number (r(2) = .995). The average diameter of a 250-cell aggregate was 95 +/- 8 microm (mean +/- SD) compared with 122 +/- 46 microm of freshly isolated islets. Islet cell loss may be minimized by performing reaggregation in the presence of medium glucose (11 mmol/L) and the GLP-1 analogue Exendin-4. Morphology, cellular composition, and architecture of reaggregated islets were comparable to intact islets. The "hanging drop" culture method allowed us to obtain pseudoislets of standardized size and regular shape, which did not differ from intact islets in terms of cellular composition or architecture. Further investigations are required to minimize cell loss and test in vivo function of transplanted pseudoislets.

Enhancement of islet engraftment and achievement of long-term islet allograft survival by Toll-like receptor 4 blockade.

PubMed

Giovannoni, Laurianne; Muller, Yannick D; Lacotte, Stéphanie; Parnaud, Géraldine; Borot, Sophie; Meier, Raphaël P H; Lavallard, Vanessa; Bédat, Benoît; Toso, Christian; Daubeuf, Bruno; Elson, Greg; Shang, Limin; Morel, Philippe; Kosco-Vilbois, Marie; Bosco, Domenico; Berney, Thierry

2015-01-01

Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. In vitro TLR4 blockade decreased lipopolysaccharide-mediated β-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.

Role of endogenous insulin gene enhancer protein ISL-1 in angiogenesis

PubMed Central

Xiong, Si-qi; Jiang, Hai-bo; Li, Yan-xiu; Li, Hai-bo; Xu, Hui-zhuo; Wu, Zhen-kai; Zheng, Wei

2016-01-01

Objective To elucidate the role of insulin gene enhancer protein ISL-1 (Islet-1) in angiogenesis and regulation of vascular endothelial growth factor (VEGF) expression in vitro and in vivo. Methods siRNA targeting Islet-1 was transfected to human umbilical vein endothelial cell lines (HUVECs). The expression of Islet-1 and VEGF in the cultured cells was measured using real-time PCR and immunoblotting. 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide; thiazolyl blue (MTT) assay was used to analyze the proliferation of HUVECs affected by Islet-1. Wound healing and Transwell assays were conducted to assess the motility of HUVECs. The formation of capillary-like structures was examined using growth factor–reduced Matrigel. siRNA targeting Islet-1 was intravitreally injected into the murine model of oxygen-induced retinopathy (OIR). Retinal neovascularization was evaluated with angiography using fluorescein-labeled dextran and then quantified histologically. Real-time PCR and immunoblotting were used to determine whether local Islet-1 silencing affected the expression of Islet-1 and VEGF in murine retinas. Results The expression of Islet-1 and VEGF in HUVECs was knocked down by siRNA. Reduced endogenous Islet-1 levels in cultured cells greatly inhibited the proliferation, migration, and tube formation in HUVECs in vitro. Retinal neovascularization following injection of Islet-1 siRNA was significantly reduced compared with that of the contralateral control eye. Histological analysis indicated that the neovascular nuclei protruding into the vitreous cavity were decreased. Furthermore, the Islet-1 and VEGF expression levels were downregulated in murine retinas treated with siRNA against Islet-1. Conclusions Reducing the expression of endogenous Islet-1 inhibits proliferation, migration, and tube formation in vascular endothelial cells in vitro and suppresses retinal angiogenesis in vivo. Endogenous Islet-1 regulates angiogenesis via VEGF. PMID:27994436

Microwell Scaffolds for the Extrahepatic Transplantation of Islets of Langerhans

PubMed Central

Buitinga, Mijke; Truckenmüller, Roman; Engelse, Marten A.; Moroni, Lorenzo; Ten Hoopen, Hetty W. M.; van Blitterswijk, Clemens A.; de Koning, Eelco JP.; van Apeldoorn, Aart A.; Karperien, Marcel

2013-01-01

Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT) block copolymer (composition: 4000PEOT30PBT70) and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet’s native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation. PMID:23737999

A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

PubMed Central

Hayes, Heather L.; Zhang, Lu; Becker, Thomas C.; Haldeman, Jonathan M.; Stephens, Samuel B.; Arlotto, Michelle; Moss, Larry G.; Newgard, Christopher B.

2016-01-01

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in β-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a β-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and β cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor β (TGF-β) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and β-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and β cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation. PMID:27620967

Engineering of microscale three-dimensional pancreatic islet models in vitro and their biomedical applications.

PubMed

Gao, Bin; Wang, Lin; Han, Shuang; Pingguan-Murphy, Belinda; Zhang, Xiaohui; Xu, Feng

2016-08-01

Diabetes now is the most common chronic disease in the world inducing heavy burden for the people's health. Based on this, diabetes research such as islet function has become a hot topic in medical institutes of the world. Today, in medical institutes, the conventional experiment platform in vitro is monolayer cell culture. However, with the development of micro- and nano-technologies, several microengineering methods have been developed to fabricate three-dimensional (3D) islet models in vitro which can better mimic the islet of pancreases in vivo. These in vitro islet models have shown better cell function than monolayer cells, indicating their great potential as better experimental platforms to elucidate islet behaviors under both physiological and pathological conditions, such as the molecular mechanisms of diabetes and clinical islet transplantation. In this review, we present the state-of-the-art advances in the microengineering methods for fabricating microscale islet models in vitro. We hope this will help researchers to better understand the progress in the engineering 3D islet models and their biomedical applications such as drug screening and islet transplantation.

Culture of impure human islet fractions in the presence of alpha-1 antitrypsin prevents insulin cleavage and improves islet recovery.

PubMed

Loganathan, G; Dawra, R K; Pugazhenthi, S; Wiseman, A C; Sanders, M A; Saluja, A K; Sutherland, D E R; Hering, B J; Balamurugan, A N

2010-01-01

Exocrine tissue is commonly cotransplanted with islets in autografting and allotransplantation of impure preparations. Proteases and insulin are released by acinar cells and islets, respectively, during pretransplantation culture and also systemically after transplantation. We hypothesized that released proteases could cleave insulin molecules and that addition of alpha-1 antitrypsin (A1AT) to impure islet cultures would block this cleavage, improving islet recovery and function. Trypsin, chymotrypsin, and elastase (TCE) activity and insulin levels were measured in culture supernates of pure (n = 5) and impure (n = 5) islet fractions, which were isolated from deceased donors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect insulin after incubation with proteases. We assessed the effects of A1AT supplementation (0.5 mg/mL; n = 4] on TCE activity, insulin levels, culture recovery, and islet quality. The ultrastructure of islets exposed to TCE versus control medium was examined using electron microscopy (EM). Protease (TCE) activity in culture supernatants was indirectly proportional to the percentage purity of islets: pure, impure, or highly impure. Increasingly lower levels of insulin were detected in culture supernatants when higher protease activity levels were present. Insulin levels measured from supernatants of impure and highly impure islet preparations were 61 +/- 23.7% and 34 +/- 33% of that in pure preparations, respectively. Incubation with commercially available proteases (TCE) or exocrine acinar cell supernatant cleaved insulin molecules as assessed using SDS-PAGE. Addition of A1AT to impure islet preparations reduced protease activity and restored normal insulin levels as detected using enzyme-linked immunosorbent assay (ELISA) and SDS-PAGE of culture supernates. A1AT improved insulin levels to 98% +/- 1.3% in impure and 78% +/- 34.2% in highly impure fractions compared with pure islet fractions. A1AT supplementation improved postculture recovery of islets in impure preparations compared with nontreated controls (72% +/- 9% vs 47% +/- 15%). Islet viability as measured using membrane integrity assays was similar in both the control (98% +/- 2%) and the A1AT-treated groups (99% +/- 1%). EM results revealed a reduction or absence of secretory granules after exposure to proteases (TCE). Culture of impure human islet fractions in the presence of A1AT prevented insulin cleavage and improved islet recovery. A1AT supplementation of islet culture media, therefore, may increase the proportion of human islet products that meet release criteria for transplantation. Copyright 2010 Elsevier Inc. All rights reserved.

Classification of microscopy images of Langerhans islets

NASA Astrophysics Data System (ADS)

Å vihlík, Jan; Kybic, Jan; Habart, David; Berková, Zuzana; Girman, Peter; Kříž, Jan; Zacharovová, Klára

2014-03-01

Evaluation of images of Langerhans islets is a crucial procedure for planning an islet transplantation, which is a promising diabetes treatment. This paper deals with segmentation of microscopy images of Langerhans islets and evaluation of islet parameters such as area, diameter, or volume (IE). For all the available images, the ground truth and the islet parameters were independently evaluated by four medical experts. We use a pixelwise linear classifier (perceptron algorithm) and SVM (support vector machine) for image segmentation. The volume is estimated based on circle or ellipse fitting to individual islets. The segmentations were compared with the corresponding ground truth. Quantitative islet parameters were also evaluated and compared with parameters given by medical experts. We can conclude that accuracy of the presented fully automatic algorithm is fully comparable with medical experts.

Current Status of Islet Cell Transplantation

PubMed Central

Ichii, Hirohito; Ricordi, Camillo

2013-01-01

Despite substantial advances in islet isolation methods and immunosuppressive protocol, pancreatic islet cell transplantation remains an experimental procedure currently limited to the most severe cases of type 1 diabetes mellitus (T1DM). The objectives of this treatment are to prevent severe hypoglycemic episodes in patients with hypoglycemia unawareness, and to achieve a more physiological metabolic control. Insulin independence and long term-graft function with improvement of quality of life have been obtained in several international islet transplant centers. However, experimental trials of islet transplantation clearly highlighted several obstacles that remain to be overcome before the procedure could be proposed to a much larger patient population. This review provides a brief historical perspective of islet transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, and outlines current challenges and future directions in clinical islet transplantation. PMID:19110649

Achievement of insulin independence in three consecutive type-1 diabetic patients via pancreatic islet transplantation using islets isolated at a remote islet isolation center.

PubMed

Goss, John A; Schock, Angela P; Brunicardi, F Charles; Goodpastor, Sarah E; Garber, Alan J; Soltes, George; Barth, Merle; Froud, Tatiana; Alejandro, Rodolfo; Ricordi, Camillo

2002-12-27

As a result of advances in both immunosuppressive protocols and pancreatic islet isolation techniques, insulin independence has recently been achieved in several patients with type 1 diabetes mellitus via pancreatic islet transplantation (PIT). Although the dissemination of immunosuppressive protocols is quite easy, transferring the knowledge and expertise required to isolate a large number of quality human islets for transplantation is a far greater challenge. Therefore, in an attempt to centralize the critical islet processing needed for islet transplantation and to avoid the development of another islet processing center, we have established a collaborative islet transplant program between two geographically distant transplant centers. Three consecutive patients with type 1 diabetes mellitus with a history of severe hypoglycemia and metabolic instability underwent PIT at the Methodist Hospital (TMH), Houston, Texas, using pancreatic islets. All pancreatic islets were isolated from pancreata procured in Houston and subsequently transported for isolation to the Human Islet Cell Processing Facility of the Diabetes Research Institute (DRI) at the University of Miami, Miami, Florida. Pancreatic islets were isolated at DRI after enzymatic ductal perfusion (Liberase-HI) by the automated method (Ricordi Chamber) using endotoxin-free and xenoprotein-free media. After purification, the islets were immediately transported back to TMH and transplanted via percutaneous transhepatic portal embolization. Immunosuppression consisted of sirolimus, tacrolimus, and daclizumab. After donor cross-clamp in Houston, donor pancreata arrived at DRI and the isolation process began within 6.5 hr in all cases (median, 5.4 hr; range, 4.8-6.5 hr). At the completion of the isolation process, the islets were immediately transported back to TMH and transplanted. All three patients attained sustained insulin independence after transplantation of 395,567, 394,381, and 563,206 pancreatic islet equivalents (IEQ), respectively. Despite insulin independence, the first two patients received less than 10,000 IEQ/kg; therefore, to increase their functional pancreatic islet reserve, they underwent a second islet transplant with 326,720 and 768,132 IEQ, respectively. Posttransplantation follow-up for these three patients is 4, 3, and 0.5 months, respectively. The mean glycosylated hemoglobin values have been dramatically reduced in the first two patients. In addition, the mean amplitude of glycemic excursions have also been reduced in all three recipients (patient 1: before transplantation 197 mg/dL vs. after transplantation 61 mg/dL; patient 2: before transplantation 202 mg/dL vs. after transplantation 52 mg/dL; patient 3: before transplantation 245 mg/dL vs. after transplantation 58 mg/dL) after PIT. All pancreatic islet allografts demonstrated the ability to respond to an in vitro glucose stimulus at the DRI before shipment and at TMH after shipment and final processing with a median stimulation index of 2.1 and 2.2, respectively. None of the transplant recipients have had a hyper- or hypoglycemic episode since PIT and no complications have occurred. These early data demonstrate that (1) pancreatic islets remain viable after shipment to remote transplant sites; (2) pancreatic islet isolation techniques and experience can be concentrated at a small number of regional facilities that could supply islets to remote transplant centers; and (3) insulin independence via PIT can be achieved using a remote pancreatic islet isolation center.

Application of Digital Image Analysis to Determine Pancreatic Islet Mass and Purity in Clinical Islet Isolation and Transplantation

PubMed Central

Wang, Ling-jia; Kissler, Hermann J; Wang, Xiaojun; Cochet, Olivia; Krzystyniak, Adam; Misawa, Ryosuke; Golab, Karolina; Tibudan, Martin; Grzanka, Jakub; Savari, Omid; Grose, Randall; Kaufman, Dixon B; Millis, Michael; Witkowski, Piotr

2015-01-01

Pancreatic islet mass, represented by islet equivalent (IEQ), is the most important parameter in decision making for clinical islet transplantation. To obtain IEQ, the sample of islets is routinely counted manually under a microscope and discarded thereafter. Islet purity, another parameter in islet processing, is routinely acquired by estimation only. In this study, we validated our digital image analysis (DIA) system developed using the software of Image Pro Plus for islet mass and purity assessment. Application of the DIA allows to better comply with current good manufacturing practice (cGMP) standards. Human islet samples were captured as calibrated digital images for the permanent record. Five trained technicians participated in determination of IEQ and purity by manual counting method and DIA. IEQ count showed statistically significant correlations between the manual method and DIA in all sample comparisons (r >0.819 and p < 0.0001). Statistically significant difference in IEQ between both methods was found only in High purity 100μL sample group (p = 0.029). As far as purity determination, statistically significant differences between manual assessment and DIA measurement was found in High and Low purity 100μL samples (p<0.005), In addition, islet particle number (IPN) and the IEQ/IPN ratio did not differ statistically between manual counting method and DIA. In conclusion, the DIA used in this study is a reliable technique in determination of IEQ and purity. Islet sample preserved as a digital image and results produced by DIA can be permanently stored for verification, technical training and islet information exchange between different islet centers. Therefore, DIA complies better with cGMP requirements than the manual counting method. We propose DIA as a quality control tool to supplement the established standard manual method for islets counting and purity estimation. PMID:24806436

That which does not kill us makes us stronger--does Nietzsche's quote apply to islets? A re-evaluation of the passenger leukocyte theory, free radicals, and glucose toxicity in islet cell transplantation.

PubMed

Wright, J R; Xu, B-Y

2014-07-01

In clinical islet transplantation, isolated islets are embolized into the liver via the portal vein (PV); however, up to 70% of the islets are lost in the first few days after transplantation (i.e., too quickly to be mediated by the adaptive immune system). Part of early loss is due to instant blood-mediated inflammatory reaction, an immune/thrombotic process caused by islets interacting with complement. We have shown that glucose toxicity (GT) also plays a critical role based upon the observation that islets embolized into the PVs of diabetic athymic mice are rapidly lost but, if recipients are not diabetic, the islet grafts persist. Using donor islets resistant to the β-cell toxin streptozotocin, we have shown that intraportal islets engrafted in non-diabetic athymic mice for as little as 3 days will maintain normoglycemia when streptozotocin is administered destroying the recipient's native pancreas β-cells. What is the mechanism of GT in β-cells? Chronic exposure to hyperglycemia over-exerts β-cells and their electron transport chains leak superoxide radicals during aerobic metabolism. Here we reinterpret old data and present some compelling new data supporting a new model of early intraportal islet graft loss. We hypothesize that diabetes stimulates overproduction of superoxide in both the β-cells of the islet grafts and the endothelial cells lining the intraportal microvasculature adjacent to where the embolized islets become lodged. This double dose of oxidant damage stresses both the islets, which are highly susceptible to free radicals because of inherent low levels of scavenging enzymes, and the adjacent hepatic endothelial cells. This, superimposed upon localized endothelial damage caused by embolization, precipitates inflammation and coagulation which further damages islet grafts. Based upon this model, we predict that pre-exposing islets to sub-lethal hyperoxia should up-regulate islet free radical scavenging enzyme levels and promote initial engraftment; reinterpretation of 30 years old "passenger leukocyte" data and preliminary new data support this. Other data suggests that pre-exposure of recipients to hyperoxia could up-regulate antioxidant enzymes in the hepatic endothelium. The combination of both effects could markedly enhance early intraportal islet graft survival and engraftment. Finally, if our model is correct, current in vitro and in vivo tests used to test batches of harvested islets for viability and function prior to transplantation are poorly conceived (n.b., it is already well-known that results using these tests often do not predict clinical islet transplantation success) and a different testing paradigm is suggested. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bioengineering a highly vascularized matrix for the ectopic transplantation of islets

PubMed Central

Ellis, Cara E; Suuronen, Erik; Yeung, Telford; Seeberger, Karen; Korbutt, Gregory S

2013-01-01

Islet transplantation is a promising treatment for Type 1 diabetes; however limitations of the intra-portal site and poor revascularization of islets must be overcome. We hypothesize that engineering a highly vascularized collagen-based construct will allow islet graft survival and function in alternative sites. In this study, we developed such a collagen-based biomaterial. Neonatal porcine islets (NPIs) were embedded in collagen matrices crosslinked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide containing combinations of chondroitin-6-sulfate, chitosan, and laminin, and compared with controls cultured in standard media. Islets were examined for insulin secretory activity after 24 h and 4 d and for apoptotic cell death and matrix integrity after 7 d in vitro. These same NPI/collagen constructs were transplanted subcutaneously in immunoincompetent B6.Rag−/− mice and then assessed for islet survival and vascularization. At all time points assessed during in vitro culture there were no significant differences in insulin secretory activity between control islets and those embedded in the collagen constructs, indicating that the collagen matrix had no adverse effect on islet function. Less cell death was observed in the matrix with all co-polymers compared with the other matrices tested. Immunohistochemical analysis of the grafts post-transplant confirmed the presence of intact insulin-positive islets; grafts were also shown to be vascularized by von Willebrand factor staining. This study demonstrates that a collagen, chondroitin-6-sulfate, chitosan, and laminin matrix supports islet function in vitro and moreover allows islet survival and vascularization post-transplantation; therefore, this bio-engineered vascularized construct is capable of supporting islet survival. PMID:24262950

An Isolated Venous Sac as a Novel Site for Cell Therapy in Diabetes Mellitus

PubMed Central

Kakabadze, Zurab; Shanava, Koba; Ricordi, Camillo; Shapiro, A.M. James; Gupta, Sanjeev; Berishvili, Ekaterine

2013-01-01

Background Transplanting pancreatic islets is of significant interest for type 1 diabetes mellitus. After intraportal injection of islets, inferior engraftment and eventual loss of transplanted islets constitute major limitations. Therefore, alternative approaches will be helpful. Here, we evaluated in animals whether an isolated venous sac would support survival of transplanted islets, along with correction of hyperglycemia. Methods Pancreatic islets isolated from adult Lewis rats were transplanted either into an isolated venous sac made from lumbar vein or into the portal vein of syngeneic rats. The integrity and vascular organization of the venous sac was determined by studies of the local microcirculation. The engraftment, survival, and function of transplanted islets were analyzed by histology, including endocrine function in situ and by glycemic control in rats with streptozotocin-induced diabetes. Results Transplanted islets showed normal morphology with insulin expression in isolated venous sac during the long term. Transplanted islets received blood supply from vasa vasorum and had access to drainage through venous tributaries in the venous sac. This resulted in restoration of euglycemia in diabetic rats. Removal of islet graft-bearing venous sac in diabetic rats led to recurrence of hyperglycemia. By contrast, euglycemia was not restored in rats treated by intraportal transplantation of islets. Conclusions We demonstrated that pancreatic islets successfully engrafted and functioned in the isolated venous sac with ability to restore euglycemia in diabetic rats. Therefore, the isolated venous sac offers a new site for transplantation of pancreatic islets. This would be clinically beneficial as an alternative to intrahepatic islet transplantation. PMID:22814331

Pancreas preservation for pancreas and islet transplantation

PubMed Central

Iwanaga, Yasuhiro; Sutherland, David E.R.; Harmon, James V.; Papas, Klearchos K.

2010-01-01

Purpose of review To summarize advances and limitations in pancreas procurement and preservation for pancreas and islet transplantation, and review advances in islet protection and preservation. Recent findings Pancreases procured after cardiac death, with in-situ regional organ cooling, have been successfully used for islet transplantation. Colloid-free Celsior and histidine-tryptophan-ketoglutarate preservation solutions are comparable to University of Wisconsin solution when used for cold storage before pancreas transplantation. Colloid-free preservation solutions are inferior to University of Wisconsin solution for pancreas preservation prior to islet isolation and transplantation. Clinical reports on pancreas and islet transplants suggest that the two-layer method may not offer significant benefits over cold storage with the University of Wisconsin solution: improved oxygenation may depend on the graft size; benefits in experimental models may not translate to human organs. Improvements in islet yield and quality occurred from pancreases treated with inhibitors of stress-induced apoptosis during procurement, storage, isolation or culture. Pancreas perfusion may be desirable before islet isolation and transplantation and may improve islet yields and quality. Methods for real-time, noninvasive assessment of pancreas quality during preservation have been implemented and objective islet potency assays have been developed and validated. These innovations should contribute to objective evaluation and establishment of improved pancreas preservation and islet isolation strategies. Summary Cold storage may be adequate for preservation before pancreas transplants, but insufficient when pancreases are processed for islets or when expanded donors are used. Supplementation of cold storage solutions with cytoprotective agents and perfusion may improve pancreas and islet transplant outcomes. PMID:18685343

Islet cell transplantation today.

PubMed

Bretzel, Reinhard G; Jahr, Henning; Eckhard, Michael; Martin, Isabel; Winter, Daniel; Brendel, Mathias D

2007-05-01

Long-term studies strongly suggest that tight control of blood glucose can prevent the development and retard the progression of chronic complications of type 1 diabetes mellitus. In contrast to conventional insulin treatment, replacement of a patient's islets of Langerhans either by pancreas organ transplantation or by isolated islet transplantation is the only treatment to achieve a constant normoglycemic state and avoiding hypoglycemic episodes, a typical adverse event of multiple daily insulin injections. However, the cost of this benefit is still the need for immunosuppressive treatment of the recipient with all its potential risks. Islet cell transplantation offers the advantage of being performed as a minimally invasive procedure in which islets can be perfused percutaneously into the liver via the portal vein. Between January 1990 and December 2004, 458 pancreatic islet transplants worldwide have been reported to the International Islet Transplant Registry (ITR) at our Third Medical Department, University of Giessen/Germany. Data analysis of islet cell transplants performed in the last 5 years (1999-2004) shows at 1 year after adult islet transplantation a patient survival rate of 97%, a functioning islet graft in 82% of the cases, whereas insulin independence was meanwhile achieved in 43% of the cases. However, using a novel protocol established by the Edmonton Center/Canada, the insulin independence rates have improved significantly reaching meanwhile a 50-80% level. Finally, the concept of islet cell or stem cell transplantation is most attractive, as it offers many perspectives: islet cell availability could become unlimited and islet or stem cells my be transplanted without life-long immunosuppressive treatment of the recipient, just to mention two of them.

Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome

PubMed Central

Griffiths, Lisa A.; Persaud, Shanta J.; Jones, Peter M.; Howell, Simon L.; Welsh, Nils

2016-01-01

Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome. Methods Islets were isolated from C57Bl/6 mice and pre-cultured with imatinib prior to exposure to streptozotocin and cytokines in vitro. Cell viability and glucose-induced insulin secretion were measured. For transplantation experiments, islets were pre-cultured with imatinib for either 72 h or 24 h prior to transplantation into streptozotocin-diabetic C57Bl/6 mice. In one experimental series mice were also administered imatinib after islet transplantation. Results Imatinib partially protected islets from beta cell death in vitro. However, pre-culturing islets in imatinib or administering the drug to the mice in the days following islet transplantation did not improve blood glucose concentrations more than control-cultured islets. Conclusion Although imatinib protected against beta cell death from cytokines and streptozotocin in vitro, it did not significantly improve syngeneic islet transplantation outcome. PMID:26953716

Experimental studies on islets isolation, purification and function in rats

PubMed Central

Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

2015-01-01

To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient. PMID:26885021

The Spleen Is an Ideal Site for Inducing Transplanted Islet Graft Expansion in Mice

PubMed Central

Takahashi, Hiroyuki; Kodama, Shohta

2017-01-01

Alternative islet transplantation sites have the potential to reduce the marginal number of islets required to ameliorate hyperglycemia in recipients with diabetes. Previously, we reported that T cell leukemia homeobox 1 (Tlx1)+ stem cells in the spleen effectively regenerated into insulin-producing cells in the pancreas of non-obese diabetic mice with end-stage disease. Thus, we investigated the spleen as a potential alternative islet transplantation site. Streptozotocin-induced diabetic C57BL/6 mice received syngeneic islets into the portal vein (PV), beneath the kidney capsule (KC), or into the spleen (SP). The marginal number of islets by PV, KC, or SP was 200, 100, and 50, respectively. Some plasma inflammatory cytokine levels in the SP group were significantly lower than those of the PV group after receiving a marginal number of islets, indicating reduced inflammation in the SP group. Insulin contents were increased 280 days after islet transplantation compared with those immediately following transplantation (p<0.05). Additionally, Tlx1-related genes, including Rrm2b and Pla2g2d, were up-regulated, which indicates that islet grafts expanded in the spleen. The spleen is an ideal candidate for an alternative islet transplantation site because of the resulting reduced inflammation and expansion of the islet graft. PMID:28135283

Imatinib prevents beta cell death in vitro but does not improve islet transplantation outcome.

PubMed

King, Aileen J F; Griffiths, Lisa A; Persaud, Shanta J; Jones, Peter M; Howell, Simon L; Welsh, Nils

2016-05-01

Introduction Improving islet transplantation outcome could not only bring benefits to individual patients but also widen the patient pool to which this life-changing treatment is available. Imatinib has previously been shown to protect beta cells from apoptosis in a variety of in vitro and in vivo models. The aim of this study was to investigate whether imatinib could be used to improve islet transplantation outcome. Methods Islets were isolated from C57Bl/6 mice and pre-cultured with imatinib prior to exposure to streptozotocin and cytokines in vitro. Cell viability and glucose-induced insulin secretion were measured. For transplantation experiments, islets were pre-cultured with imatinib for either 72 h or 24 h prior to transplantation into streptozotocin-diabetic C57Bl/6 mice. In one experimental series mice were also administered imatinib after islet transplantation. Results Imatinib partially protected islets from beta cell death in vitro. However, pre-culturing islets in imatinib or administering the drug to the mice in the days following islet transplantation did not improve blood glucose concentrations more than control-cultured islets. Conclusion Although imatinib protected against beta cell death from cytokines and streptozotocin in vitro, it did not significantly improve syngeneic islet transplantation outcome.

Microfluidic platform for assessing pancreatic islet functionality through dielectric spectroscopy

PubMed Central

Heileman, K.; Daoud, J.; Hasilo, C.; Gasparrini, M.; Paraskevas, S.; Tabrizian, M.

2015-01-01

Human pancreatic islets are seldom assessed for dynamic responses to external stimuli. Thus, the elucidation of human islet functionality would provide insights into the progression of diabetes mellitus, evaluation of preparations for clinical transplantation, as well as for the development of novel therapeutics. The objective of this study was to develop a microfluidic platform for in vitro islet culture, allowing the multi-parametric investigation of islet response to chemical and biochemical stimuli. This was accomplished through the fabrication and implementation of a microfluidic platform that allowed the perifusion of islet culture while integrating real-time monitoring using impedance spectroscopy, through microfabricated, interdigitated electrodes located along the microchamber arrays. Real-time impedance measurements provide important dielectric parameters, such as cell membrane capacitance and cytoplasmic conductivity, representing proliferation, differentiation, viability, and functionality. The perifusion of varying glucose concentrations and monitoring of the resulting impedance of pancreatic islets were performed as proof-of-concept validation of the lab-on-chip platform. This novel technique to elucidate the underlying mechanisms that dictate islet functionality is presented, providing new information regarding islet function that could improve the evaluation of islet preparations for transplantation. In addition, it will lead to a better understanding of fundamental diabetes-related islet dysfunction and the development of therapeutics through evaluation of potential drug effects. PMID:26339324

Islet autotransplantation to prevent or minimize diabetes after pancreatectomy.

PubMed

Carlson, Annelisa M; Kobayashi, Takashi; Sutherland, David Er

2007-02-01

Islet autotransplantation can prevent or minimize diabetes following near or total pancreatectomy for chronic pancreatitis or other lesions. Since the first case nearly 30 years ago, islet autotransplantation has been performed at more than 20 centers. This review summarizes outcomes and factors that correlate with success or failure. The main criteria for success of an islet autotransplantation per se are whether insulin-independence was maintained or insulin-need minimized, but, for those with chronic pancreatitis, as important is the degree of pain reduction, narcotic withdrawal, and quality of life improvement. Total pancreatectomy/islet autotransplantation for chronic pancreatitis usually ameliorates pain and improves quality of life. The higher the islet yield, the more likely is the patient to be insulin-independent or metabolically stable. A prior Puestow procedure or distal pancreatectomy, or long-standing disease with severe pancreatic fibrosis, predisposes to poor islet yield. In recipients who require insulin, β cell function facilitates glycemic control. Islet autotransplantation function for more than a decade has been documented, but more studies are needed to determine durability. Islet autotransplantation preserves β cell function after total pancreatectomy. Future studies comparing function of islet autografts and allografts matched for initial β cell mass may help determine the immunological and nonimmunological factors that influence long-term islet survival.

A macroporous heparin-releasing silk fibroin scaffold improves islet transplantation outcome by promoting islet revascularisation and survival.

PubMed

Mao, Duo; Zhu, Meifeng; Zhang, Xiuyuan; Ma, Rong; Yang, Xiaoqing; Ke, Tingyu; Wang, Lianyong; Li, Zongjin; Kong, Deling; Li, Chen

2017-09-01

Islet transplantation is considered the most promising therapeutic option with the potential to cure diabetes. However, efficacy of current clinical islet transplantation is limited by long-term graft dysfunction and attrition. We have investigated the therapeutic potential of a silk fibroin macroporous (SF) scaffold for syngeneic islet transplantation in diabetic mice. The SF scaffold was prepared via lyophilisation, which enables incorporation of active compounds including cytokines, peptide and growth factors without compromising their biological activity. For the present study, a heparin-releasing SF scaffold (H-SF) in order to evaluate the versatility of the SF scaffold for biological functionalisation. Islets were then co-transplanted with H-SF or SF scaffolds in the epididymal fat pad of diabetic mice. Mice from both H-SF and SF groups achieved 100% euglycaemia, which was maintained for 1year. More importantly, the H-SF-islets co-transplantation led to more rapid reversal of hyperglycaemia, complete normalisation of glucose responsiveness and lower long-term blood glucose levels. This superior transplantation outcome is attributable to H-SF-facilitated islet revascularisation and cell proliferation since significant increase of islet endocrine and endothelial cells proliferation was shown in grafts retrieved from H-SF-islets co-transplanted mice. Better intra-islet vascular reformation was also evident, accompanied by VEGF upregulation. In addition, when H-SF was co-transplanted with islets extracted from vegfr2-luc transgenic mice in vivo, sustained elevation of bioluminescent signal that corresponds to vegfr2 expression was collected, implicating a role of heparin-dependent activation of endogenous VEGF/VEGFR2 pathway in promoting islet revascularisation and proliferation. In summary, the SF scaffolds provide an open platform as scaffold development for islet transplantation. Furthermore, given the pro-angiogenic, pro-survival and minimal post-transplantation inflammatory reactions of H-SF, our data also support the feasibility of clinical implementation of H-SF to improve islet transplantation outcome. 1) The silk fibroin scaffold presented in the present study provides an open platform for scaffold development in islet transplantation, with heparinisation as an example. 2) Both heparin and silk fibroin have been used clinically. The excellent in vivo therapeutic outcome reported here may therefore be clinically relevant and provide valuable insights for bench to bed translation. 3) Compared to conventional clinical islet transplantation, during which islets are injected via the hepatic portal vein, the physical/mechanical properties of silk fibroin scaffolds create a more accessible transplantation site (i.e., within fat pad), which significantly reduces discomfort. 4) Islet implantation into the fat pad also avoids an instant blood mediated inflammatory response, which occurs upon contact of islet with recipient's blood during intraportal injection, and prolongs survival and function of implanted islets. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

3-D Imaging Reveals Participation of Donor Islet Schwann Cells and Pericytes in Islet Transplantation and Graft Neurovascular Regeneration.

PubMed

Juang, Jyuhn-Huarng; Kuo, Chien-Hung; Peng, Shih-Jung; Tang, Shiue-Cheng

2015-02-01

The primary cells that participate in islet transplantation are the endocrine cells. However, in the islet microenvironment, the endocrine cells are closely associated with the neurovascular tissues consisting of the Schwann cells and pericytes, which form sheaths/barriers at the islet exterior and interior borders. The two cell types have shown their plasticity in islet injury, but their roles in transplantation remain unclear. In this research, we applied 3-dimensional neurovascular histology with cell tracing to reveal the participation of Schwann cells and pericytes in mouse islet transplantation. Longitudinal studies of the grafts under the kidney capsule identify that the donor Schwann cells and pericytes re-associate with the engrafted islets at the peri-graft and perivascular domains, respectively, indicating their adaptability in transplantation. Based on the morphological proximity and cellular reactivity, we propose that the new islet microenvironment should include the peri-graft Schwann cell sheath and perivascular pericytes as an integral part of the new tissue.

The Current Status of Islet Transplantation and its Perspectives

PubMed Central

Kobayashi, Naoya

2008-01-01

Transplantation of human pancreatic isolated islets can restore beta-cell function but it requires chronic immunosuppression. The outcome of islet transplantation mainly depends on both the quality of islet preparations, and the survival of the graft. The quality of islet preparations can be evaluated by the results of isolation, which determines the chance to achieve insulin independence. The survival of islet grafts is reflected by the amount of engrafted functional tissue that maintains metabolic control. Immunosuppressive therapy prevents the immunological rejection of grafts, but impairs their function and impedes their regenerative capacity. Therefore, the selection of high quality islet preparations and the reduction of toxic effects of immunosuppressive regimens might dramatically improve the outcomes. The application of stem cell therapy in islet transplantation may contribute to a better understanding of the mechanisms responsible for tissue homeostasis and immune tolerance. Xenogeneic islets may serve as an unlimited source if immune tolerance can be achieved. This may be a strategy to enable a substantial improvement in function while overcoming potentially deleterious risks. PMID:19099085

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation.

PubMed

Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

2007-01-03

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called beta cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding beta-cell function at the molecular level will likely facilitate the development of techniques to manufacture beta-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release.

Pancreatic islet cell therapy for type I diabetes: understanding the effects of glucose stimulation on islets in order to produce better islets for transplantation

PubMed Central

Ren, Jiaqiang; Jin, Ping; Wang, Ena; Liu, Eric; Harlan, David M; Li, Xin; Stroncek, David F

2007-01-01

While insulin replacement remains the cornerstone treatment for type I diabetes mellitus (T1DM), the transplantation of pancreatic islets of Langerhans has the potential to become an important alternative. And yet, islet transplant therapy is limited by several factors, including far too few donor pancreases. Attempts to expand mature islets or to produce islets from stem cells are far from clinical application. The production and expansion of the insulin-producing cells within the islet (so called β cells), or even creating cells that secrete insulin under appropriate physiological control, has proven difficult. The difficulty is explained, in part, because insulin synthesis and release is complex, unique, and not entirely characterized. Understanding β-cell function at the molecular level will likely facilitate the development of techniques to manufacture β-cells from stem cells. We will review islet transplantation, as well as the mechanisms underlying insulin transcription, translation and glucose stimulated insulin release. PMID:17201925

Extracellular Matrix Scaffold Technology for Bioartificial Pancreas Engineering

PubMed Central

Salvatori, Marcus; Katari, Ravi; Patel, Timil; Peloso, Andrea; Mugweru, Jon; Owusu, Kofi

2014-01-01

Emergent technologies in regenerative medicine may soon overcome the limitations of conventional diabetes therapies. Collaborative efforts across the subfields of stem cell technology, islet encapsulation, and biomaterial carriers seek to produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes. These technologies rely on a robust understanding of the extracellular matrix (ECM), the supportive 3-dimensional network of proteins necessary for cellular attachment, proliferation, and differentiation. Although these functions can be partially approximated by biosynthetic carriers, novel decellularization protocols have allowed researchers to discover the advantages afforded by the native pancreatic ECM. The native ECM has proven to be an optimal platform for recellularization and whole-organ pancreas bioengineering, an exciting new field with the potential to resolve the dire shortage of transplantable organs. This review seeks to contextualize recent findings, discuss current research goals, and identify future challenges of regenerative medicine as it applies to diabetes management. PMID:24876552

Placental insufficiency decreases pancreatic vascularity and disrupts hepatocyte growth factor signaling in the pancreatic islet endothelial cell in fetal sheep.

PubMed

Rozance, Paul J; Anderson, Miranda; Martinez, Marina; Fahy, Anna; Macko, Antoni R; Kailey, Jenai; Seedorf, Gregory J; Abman, Steven H; Hay, William W; Limesand, Sean W

2015-02-01

Hepatocyte growth factor (HGF) and vascular endothelial growth factor A (VEGFA) are paracrine hormones that mediate communication between pancreatic islet endothelial cells (ECs) and β-cells. Our objective was to determine the impact of intrauterine growth restriction (IUGR) on pancreatic vascularity and paracrine signaling between the EC and β-cell. Vessel density was less in IUGR pancreata than in controls. HGF concentrations were also lower in islet EC-conditioned media (ECCM) from IUGR, and islets incubated with control islet ECCM responded by increasing insulin content, which was absent with IUGR ECCM. The effect of ECCM on islet insulin content was blocked with an inhibitory anti-HGF antibody. The HGF receptor was not different between control and IUGR islets, but VEGFA was lower and the high-affinity VEGF receptor was higher in IUGR islets and ECs, respectively. These findings show that paracrine actions from ECs increase islet insulin content, and in IUGR ECs, secretion of HGF was diminished. Given the potential feed-forward regulation of β-cell VEGFA and islet EC HGF, these two growth factors are highly integrated in normal pancreatic islet development, and this regulation is decreased in IUGR fetuses, resulting in lower pancreatic islet insulin concentrations and insulin secretion. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Harnessing the Foreign Body Reaction in Marginal Mass Device-less Subcutaneous Islet Transplantation in Mice.

PubMed

Pepper, Andrew R; Pawlick, Rena; Bruni, Antonio; Gala-Lopez, Boris; Wink, John; Rafiei, Yasmin; Bral, Mariusz; Abualhassan, Nasser; Shapiro, A M James

2016-07-01

Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. However, despite early insulin independence, long-term graft attrition gradually reverts recipients to exogenous insulin dependency. Undoubtedly, as insulin producing stem cell therapies progress, a transplant site that is retrievable is desirable. This prerequisite is currently incompatible with intrahepatic islet transplantation. Herein, we evaluate the functional capacity of a prevascularized subcutaneous site to accommodate marginal islet mass transplantation in mice. Syngeneic mouse islets (150) were transplanted either under the kidney capsule (KC), into a prevascularized subcutaneous device-less (DL) site, or into the unmodified subcutaneous (SC) tissue. The DL site was created 4 weeks before diabetes induction and islet transplantation through the transient placement of a 5-Fr vascular catheter. Recipient mice were monitored for glycemic control and intraperitoneal glucose tolerance. A marginal islet mass transplanted into the DL site routinely reversed diabetes (n = 13 of 18) whereas all SC islet recipients failed to restore glycemic control (n = 0 of 10, P < 0.01, log-rank). As anticipated, nearly all islet-KC mice (n = 15 of 16) became euglycemic posttransplant. The DL recipients' glucose profiles were comparable to KC islet grafts, postintrapertioneal glucose tolerance testing, whereas SC recipients remained hyperglycemic postglucose challenge. All normoglycemic mice maintained graft function for 100 days until graft retrieval. DL and KC islet grafts stained positively for insulin, microvessels, and a collagen scaffold. The device-less prevascularized approach supports marginal mass islet engraftment in mice.

Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation.

PubMed

Abualhassan, Nasser; Sapozhnikov, Lena; Pawlick, Rena L; Kahana, Meygal; Pepper, Andrew R; Bruni, Antonio; Gala-Lopez, Boris; Kin, Tatsuya; Mitrani, Eduardo; Shapiro, A M James

2016-01-01

There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.

Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation

PubMed Central

Pawlick, Rena L.; Kahana, Meygal; Pepper, Andrew R.; Bruni, Antonio; Gala-Lopez, Boris; Kin, Tatsuya; Mitrani, Eduardo; Shapiro, A. M. James

2016-01-01

There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs) made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group). Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ), 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group). Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ. PMID:27227978

Effect of the Diabetic State on Islet Engraftment and Function in a Large Animal Model of Islet–Kidney Transplantation

PubMed Central

Hirakata, Atsushi; Weiss, Matthew; Griesemer, Adam; Shimizu, Akira; Hong, Hanzhou; Habertheuer, Andreas; Tchipashvili, Vaja; Yamada, Kazuhiko; Sachs, David H.

2018-01-01

In islet transplantation, in addition to immunologic and ischemic factors, the diabetic/hyperglycemic state of the recipient has been proposed, although not yet validated, as a possible cause of islet toxicity, contributing to islet loss during the engraftment period. Using a miniature swine model of islet transplantation, we have now assessed the effect of a persistent state of hyperglycemia on islet engraftment and subsequent function. An islet–kidney (IK) model previously described by our laboratory was utilized. Three experimental donor animals underwent total pancreatectomy and autologous islet transplantation underneath the renal capsule to prepare an IK at a load of ≤1,000 islet equivalents (IE)/kg donor weight, leading to a chronic diabetic state during the engraftment period (fasting blood glucose >250 mg/dL). Three control donor animals underwent partial pancreatectomy (sufficient to maintain normoglycemia during islet engraftment period) and IK preparation. As in vivo functional readout for islet engraftment, the IKs were transplanted across an immunologic minor or class I mismatch barrier into diabetic, nephrectomized recipients at an islet load of ∼4,500 IE/kg recipient weight. A 12-d course of cyclosporine was administered for tolerance induction. All experimental donors became diabetic and showed signs of end organ injury, while control donors maintained normoglycemia. All recipients of IK from both experimental and control donors achieved glycemic control over long-term follow-up, with reversal of diabetic nephropathy and with similar glucose tolerance tests. In this preclinical, large animal model, neither islet engraftment nor subsequent long-term islet function after transplantation appear to be affected by the diabetic state. PMID:29338381

Autologous islet transplantation with remote islet isolation after pancreas resection for chronic pancreatitis.

PubMed

Tai, Denise S; Shen, Na; Szot, Gregory L; Posselt, Andrew; Feduska, Nicholas J; Habashy, Andrew; Clerkin, Barbara; Core, Erin; Busuttil, Ronald W; Hines, O Joe; Reber, Howard A; Lipshutz, Gerald S

2015-02-01

Autologous islet transplantation is an elegant and effective method for preserving euglycemia in patients undergoing near-total or total pancreatectomy for severe chronic pancreatitis. However, few centers worldwide perform this complex procedure, which requires interdisciplinary coordination and access to a sophisticated Food and Drug Administration-licensed islet-isolating facility. To investigate outcomes from a single institutional case series of near-total or total pancreatectomy and autologous islet transplantation using remote islet isolation. Retrospective cohort study between March 1, 2007, and December 31, 2013, at tertiary academic referral centers among 9 patients (age range, 13-47 years) with chronic pancreatitis and reduced quality of life after failed medical management. Pancreas resection, followed by transport to a remote facility for islet isolation using a modified Ricordi technique, with immediate transplantation via portal vein infusion. Islet yield, pain assessment, insulin requirement, costs, and transport time. Eight of nine patients had successful islet isolation after near-total or total pancreatectomy. Four of six patients with total pancreatectomy had islet yields exceeding 5000 islet equivalents per kilogram of body weight. At 2 months after surgery, all 9 patients had significantly reduced pain or were pain free. Of these patients, 2 did not require insulin, and 1 required low doses. The mean transport cost was $16,527, and the mean transport time was 3½ hours. Pancreatic resection with autologous islet transplantation for severe chronic pancreatitis is a safe and effective final alternative to ameliorate debilitating pain and to help prevent the development of surgical diabetes. Because many centers lack access to an islet-isolating facility, we describe our experience using a regional 2-center collaboration as a successful model to remotely isolate cells, with outcomes similar to those of larger case series.

HLA Class I Sensitization in Islet Transplant Recipients – Report from the Collaborative Islet Transplant Registry

PubMed Central

Naziruddin, Bashoo; Wease, Steve; Stablein, Donald; Barton, Franca B.; Berney, Thierry; Rickels, Michael R.; Alejandro, Rodolfo

2015-01-01

Pancreatic islet transplantation is a promising treatment option for patients severely affected with type 1 diabetes. This report from CITR presents pre- and post-transplant human leukocyte antigen (HLA) class I sensitization rates in islet alone transplantation. Data came from 303 recipients transplanted with islet alone between January 1999 and December 2008. HLA class I sensitization was determined by the presence of anti-HLA class I antibodies. Panel-reactive antibodies (PRA) from prior to islet infusion and at 6 months, and yearly post-transplant was correlated to measures of islet graft failure. The cumulative number of mismatched HLA alleles increased with each additional islet infusion from a median of 3 for one infusion to 9 for three infusions. Pre-transplant PRA was not predictive of islet graft failure. However, development of PRA ≥20% post-transplant was associated with 3.6 fold (p=.001) increased hazard ratio for graft failure. Patients with complete graft loss who had discontinued immunosuppression had significantly higher rate of PRA ≥ 20% compared to those with functioning grafts who remained on immunosuppression. Exposure to repeat HLA class I mismatch at second or third islet infusions resulted in less frequent development of de novo HLA class I antibodies when compared to increased class I mismatch. The development of HLA class I antibodies while on immunosuppression is associated with subsequent islet graft failure. The risk of sensitization may be reduced by minimizing the number of islet donors used per recipient, and in the absence of donor-specific anti-HLA antibodies, repeating HLA class I mismatches with subsequent islet infusions. PMID:22080832

Improvement of isolated caprine islet survival and functionality in vitro by enhancing of PDX1 gene expression.

PubMed

Hani, Homayoun; Allaudin, Zeenathul Nazariah; Mohd-Lila, Mohd-Azmi; Sarsaifi, Kazhal; Rasouli, Mina; Tam, Yew Joon; Tengku-Ibrahim, Tengku-Azmi; Othman, Abas Mazni

2017-05-01

Dead islets replaced with viable islets are a promising offer to restore normal insulin production to a person with diabetes. The main reason for establishing a new islet source for transplantation is the insufficiency of human donor pancreas while using xenogeneic islets perhaps assists this problem. The expression of PDX1 is essential for the pancreas expansion. In mature β-cells, PDX1 has several critical roles such as glucose sensing, insulin synthesis, and insulin secretion. In this study, we aimed to evaluate the expression of pancreatic duodenal homeobox-1 (PDX1) in treated caprine islets in culture and to assess the protective effects of antioxidant factors on the PDX1 gene in cultured caprine islets. Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media. Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%). Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media. © 2017 The Authors. Xenotransplantation published by John Wiley & Sons Ltd.

Diabetes Is Reversed in a Murine Model by Marginal Mass Syngeneic Islet Transplantation Using a Subcutaneous Cell Pouch Device.

PubMed

Pepper, Andrew R; Pawlick, Rena; Gala-Lopez, Boris; MacGillivary, Amanda; Mazzuca, Delfina M; White, David J G; Toleikis, Philip M; Shapiro, A M James

2015-11-01

Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation. Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance. Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels. The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies.

Evaluation of MicroRNA375 as a Novel Biomarker for Graft Damage in Clinical Islet Transplantation.

PubMed

Kanak, Mazhar A; Takita, Morihito; Shahbazov, Rauf; Lawrence, Michael C; Chung, Wen Yuan; Dennison, Ashley R; Levy, Marlon F; Naziruddin, Bashoo

2015-08-01

Early and sensitive detection of islet graft damage is essential for improving posttransplant outcomes. MicroRNA 375 (miR375) has been reported as a biomarker of pancreatic β-cell death in small animal models. The miR375 levels were measured in purified human islets, sera from patients with autologous and allogeneic islet transplantation as well as total pancreatectomy alone (nontransplanted group). The miR375 levels were also determined in a miniaturized in vitro tube model comprising human islets and autologous blood. The miR375 expression level in islets was dose-dependent (P < 0.001) and significantly elevated after islet damage in plasma in the in vitro model (P = 0.003). Clinical analysis revealed that circulating miR375 levels in both autologous and allogeneic islet recipients were significantly elevated for 7 days after islet infusion, compared with the nontransplanted group (P = 0.005 and <0.001, respectively). Furthermore, miR375 detected the difference in islet graft damage among 3 different anti-inflammatory protocols for clinical autologous transplantation (P < 0.01). Circulating miR375 can be a reliable biomarker to detect graft damage in clinical islet transplantation because serum C-peptide and proinsulin levels are difficult to interpret due to the influence of multiple factors, such as β-cell stress and physiological response.

Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets.

PubMed

Vlahos, Alexander E; Cober, Nicholas; Sefton, Michael V

2017-08-29

The transplantation of pancreatic islets, following the Edmonton Protocol, is a promising treatment for type I diabetics. However, the need for multiple donors to achieve insulin independence reflects the large loss of islets that occurs when islets are infused into the portal vein. Finding a less hostile transplantation site that is both minimally invasive and able to support a large transplant volume is necessary to advance this approach. Although the s.c. site satisfies both these criteria, the site is poorly vascularized, precluding its utility. To address this problem, we demonstrate that modular tissue engineering results in an s.c. vascularized bed that enables the transplantation of pancreatic islets. In streptozotocin-induced diabetic SCID/beige mice, the injection of 750 rat islet equivalents embedded in endothelialized collagen modules was sufficient to restore and maintain normoglycemia for 21 days; the same number of free islets was unable to affect glucose levels. Furthermore, using CLARITY, we showed that embedded islets became revascularized and integrated with the host's vasculature, a feature not seen in other s.c. Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206 + MHCII - (M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology.

Intrinsic islet heterogeneity and gap junction coupling determine spatiotemporal Ca²⁺ wave dynamics.

PubMed

Benninger, Richard K P; Hutchens, Troy; Head, W Steven; McCaughey, Michael J; Zhang, Min; Le Marchand, Sylvain J; Satin, Leslie S; Piston, David W

2014-12-02

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca(2+)]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in β-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca(2+)]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet β-cells. The extent of [Ca(2+)]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring β-cells. These results reveal how the spatiotemporal [Ca(2+)]i dynamics of the islet depend on β-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Alpha-, Delta- and PP-cells

PubMed Central

Brereton, Melissa F.; Vergari, Elisa; Zhang, Quan

2015-01-01

Islet non-β-cells, the α- δ- and pancreatic polypeptide cells (PP-cells), are important components of islet architecture and intercellular communication. In α-cells, glucagon is found in electron-dense granules; granule exocytosis is calcium-dependent via P/Q-type Ca2+-channels, which may be clustered at designated cell membrane sites. Somatostatin-containing δ-cells are neuron-like, creating a network for intra-islet communication. Somatostatin 1-28 and 1-14 have a short bioactive half-life, suggesting inhibitory action via paracrine signaling. PP-cells are the most infrequent islet cell type. The embryologically separate ventral pancreas anlage contains PP-rich islets that are morphologically diffuse and α-cell deficient. Tissue samples taken from the head region are unlikely to be representative of the whole pancreas. PP has anorexic effects on gastro-intestinal function and alters insulin and glucagon secretion. Islet architecture is disrupted in rodent diabetic models, diabetic primates and human Type 1 and Type 2 diabetes, with an increased α-cell population and relocation of non-β-cells to central areas of the islet. In diabetes, the transdifferentiation of non-β-cells, with changes in hormone content, suggests plasticity of islet cells but cellular function may be compromised. Understanding how diabetes-related disordered islet structure influences intra-islet cellular communication could clarify how non-β-cells contribute to the control of islet function. PMID:26216135

Isolation of Mouse Pancreatic Islets of Langerhans.

PubMed

Ramírez-Domínguez, Miriam

The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a "group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both" (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol.

The journey of islet cell transplantation and future development.

PubMed

Gamble, Anissa; Pepper, Andrew R; Bruni, Antonio; Shapiro, A M James

2018-03-04

Intraportal islet transplantation has proven to be efficacious in preventing severe hypoglycemia and restoring insulin independence in selected patients with type 1 diabetes. Multiple islet infusions are often required to achieve and maintain insulin independence. Many challenges remain in clinical islet transplantation, including substantial islet cell loss early and late after islet infusion. Contributions to graft loss include the instant blood-mediated inflammatory reaction, potent host auto- and alloimmune responses, and beta cell toxicity from immunosuppressive agents. Protective strategies are being tested to circumvent several of these events including exploration of alternative transplantation sites, stem cell-derived insulin producing cell therapies, co-transplantation with mesenchymal stem cells or exploration of novel immune protective agents. Herein, we provide a brief introduction and history of islet cell transplantation, limitations associated with this procedure and methods to alleviate islet cell loss as a means to improve engraftment outcomes.

Therapeutic Strategies for Modulating the Extracellular Matrix to Improve Pancreatic Islet Function and Survival After Transplantation.

PubMed

Smink, Alexandra M; de Vos, Paul

2018-05-19

Extracellular matrix (ECM) components modulate the interaction between pancreatic islet cells. During the islet isolation prior to transplantation as treatment for type 1 diabetes, the ECM is disrupted impacting functional graft survival. Recently, strategies for restoring ECM have shown to improve transplantation outcomes. This review discusses the current therapeutic strategies to modulate ECM components to improve islet engraftment. Approaches applied are seeding islets in ECM of decellularized organs, supplementation of specific ECM components in polymeric scaffolds or immunoisolating capsules, and stimulating islet ECM production with specific growth factors or ECM-producing cells. These strategies have shown success in improving functional islet survival. However, the same experiments show that caution should be taken as some ECM components may negatively impact islet function and engraftment. ECM restoration resulted in improved transplantation outcomes, but careful selection of beneficial ECM components and strategies is warranted.

Roles of Toll-like receptors in allogeneic islet transplantation.

PubMed

Ro, Han; Hong, Juho; Kim, Beom Seok; Lee, Eun Won; Kim, Myung-Gyu; Han, Kyu Hyun; Yeom, Hye-Jung; Lee, Eun Mi; Jeong, Jong Cheol; Oh, Kook-Hwan; Ahn, Curie; Yang, Jaeseok

2012-11-27

Toll-like receptors (TLRs) are involved in the rejection of solid organ allografts. However, the roles of TLRs in islets are still controversial. We investigated the roles of TLRs in donor islets together with those in recipients in allogeneic islet transplantation. To assess the roles of TLRs in either donor islets or recipients, allogeneic islet transplantation was performed using myeloid differentiation factor 88 (MyD88)-knockout (KO), TLR4-KO, or Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)-KO mice. Both polyriboinosinic polyribocytidylic acid and lipopolysaccharide (LPS) stimulation induced the mRNA expression of regulated and normal T cell expressed and secreted, interferon-γ-inducible protein-10, monocyte chemotactic protein-1, interleukin-8, and inducible nitric oxide synthase in murine islets, whereas the induction was attenuated in TRIF-KO, interferon-β promoter stimulator-1-KO, and TLR4-KO mice. When islets from MyD88-KO, TLR4-KO, or TRIF-KO C57BL/6 mice were transplanted to BALB/c recipients, graft survival was not better than that of wild-type (WT) islets. However, the survival of the MyD88-KO islet allograft was significantly prolonged when combined with anti-CD40L. In parallel, LPS stimulation in donor islets interfered with anti-CD40L blockade-mediated long-term survival of islet allografts in TLR4-KO recipients. LPS stimulation increased the perigraft infiltration of both T cells and macrophages. Then again, when islets from WT BALB/c mice were transplanted to MyD88-KO, TRIF-KO, or WT C57BL/6 mice, there was no difference in graft survival, although some of the MyD88-KO recipients obtained long-term graft survival. However, anti-CD40L prolonged graft survival significantly in MyD88-KO recipients. The absence of MyD88 in either donors or recipients decreased the perigraft infiltration of inflammatory cells when combined with anti-CD40L. TLRs in both donor islets and recipients are involved in islet allograft rejection.

Effect of Manufacturing Procedures on Human Islet Isolation from Donor Pancreata Standardized by the North American Islet Donor Score

PubMed Central

Yeh, Chun-Chieh; Wang, Ling-Jia; Mcgarrigle, James J.; Wang, Yong; Liao, Chien-Chang; Omami, Mustafa; Khan, Arshad; Nourmohammadzadeh, Mohammad; Mendoza-Elias, Joshua; Mccracken, Benjamin; Marchese, Enza; Barbaro, Barbara; Oberholzer, Jose

2017-01-01

This study investigates manufacturing procedures that affect islet isolation outcomes from donor pancreata standardized by the North American Islet Donor Score (NAIDS). Islet isolations performed at the University of Illinois, Chicago, from pancreata with NAIDS ≥65 were investigated. The research cohort was categorized into two groups based on a postpurification yield either greater than (group A) or less than (group B) 400,000 IEQ. Associations between manufacturing procedures and islet isolation outcomes were analyzed using multivariate logistic or linear regressions. A total of 119 cases were retrieved from 630 islet isolations performed since 2003. Group A is composed of 40 cases with an average postpurified yield of 570,098 IEQ, whereas group B comprised 79 cases with an average yield of 235,987 IEQ. One third of 119 cases were considered successful islet isolations that yielded >400,000 IEQ. The prepurified and postpurified islet product outcome parameters were detailed for future reference. The NAIDS (>80 vs. 65–80) [odds ratio (OR): 2.91, 95% confidence interval (CI): 1.27–6.70], cold ischemic time (≤10 vs. >10 h) (OR: 3.68, 95% CI: 1.61–8.39), and enzyme perfusion method (mechanical vs. manual) (OR: 2.38, 95% CI: 1.01–5.56) were independent determinants for postpurified islet yield ≥400,000 IEQ. The NAIDS (>80, p < 0.001), cold ischemic time (≤10 h, p < 0.05), increased unit of collagenase (p < 0.01), and pancreatic duct cannulation time (<30 min, p < 0.01) all independently correlated with better islet quantity parameters. Furthermore, cold ischemic time (≤10 h, p < 0.05), liberase MTF (p < 0.001), increased unit of collagenase (p < 0.05), duct cannulation time (<30 min, p < 0.05), and mechanical enzyme perfusion (p < 0.05) were independently associated with better islet morphology score. Analysis of islet manufacturing procedures from the pancreata with standardized quality is essential in identifying technical issues within islet isolation. Adequate processing duration in each step of islet isolation, using liberase MTF, and mechanical enzyme perfusion all affect isolation outcomes. PMID:27524672

Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

PubMed

Balamurugan, Appakalai N; Green, Michael L; Breite, Andrew G; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G; Williams, Stuart K; Hering, Bernhard J; Dwulet, Francis E; McCarthy, Robert C

2016-01-01

Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells

PubMed Central

Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang

2017-01-01

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized. PMID:28207765

A bilaminated decellularized scaffold for islet transplantation: Structure, properties and functions in diabetic mice.

PubMed

Wang, Xi; Wang, Kai; Zhang, Wei; Qiang, Ming; Luo, Ying

2017-09-01

Ectopic transplantation of islets provides a beta cell-replacement approach that may allow the recovery of physiological regulation of the blood sugar level in patients with Type I diabetes (T1D). In development of new extrahepatic islet transplantation protocols in support of the islet engraftment, it is pivotal to develop scaffold materials with multifaceted functions to provide beneficial microenvironment, mediate host response in favor of vascularization/islet integration and maintain long-term islet function at the transplantation site. In this study, a new composite bilaminar decellularized scaffold (CDS) was fabricated with differential structural, degradation and mechanical properties by the combination of a fast-degrading porous collagen matrix and a mechanically supportive porcine pericardium. When investigated in the epididymal fat pad in syngeneic mouse models, it was shown that CDS could serve as superior scaffolds to promote islet adhesion and viability, and islet-CDS constructs also allowed rapid reversal of the hyperglycemic condition in the host. The engraftment and effects of islets were achieved at low islet numbers, accompanied by minimal adverse tissue reactions and optimal islet integration with the surrounding fat tissue. The bioactive surface, mechanical/chemical durability and biocompatibility of the CDS may all have played important roles in facilitating the engraftment of islets. Our study provided new insights into scaffold's function in the interplay of cells, materials and host tissue and the extracellular matrix-based scaffolds have potential for clinical translation in the beta cell-replacement therapy to treat T1D. Copyright © 2017 Elsevier Ltd. All rights reserved.

Islet graft survival and function: concomitant culture and transplantation with vascular endothelial cells in diabetic rats.

PubMed

Pan, Xiaoming; Xue, Wujun; Li, Yang; Feng, Xinshun; Tian, Xiaohui; Ding, Chenguang

2011-12-15

Human islet transplantation is a great potential therapy for type I diabetes. To investigate islet graft survival and function, we recently showed the improved effects after co-culture and co-transplantation with vascular endothelial cells (ECs) in diabetic rats. ECs were isolated, and the viability of isolated islets was assessed in two groups (standard culture group and co-culture group with ECs). Then streptozotocin-induced diabetic rats were divided into four groups before islet transplantation as follows: group A with infusion of islet grafts; group B with combined vascular ECs and islet grafts; groups C and D as controls with single ECs infusion and phosphate-buffered saline injection, respectively. Blood glucose and insulin concentrations were measured daily. Expression of vascular endothelial growth factor was investigated by immunohistochemical staining. The mean microvascular density was also calculated. More than 90% of acridine orange-propidium iodide staining positive islets demonstrated normal morphology while co-cultured with ECs for 7 days. Compared with standard control, insulin release assays showed a significantly higher simulation index in co-culture group except for the first day (P<0.05). After transplantation, there was a significant difference in concentrations of blood glucose and insulin among these groups after 3 days (P<0.05). The mean microvascular density in co-culture group was significantly higher than that in single islet group (P=0.04). Co-culture with ECs in vitro could improve the survival and function of isolated rat islet, and co-transplantation of islets with ECs could effectively prolong the islet graft survival in diabetic rats.

Inhibition of inflammatory cytokine-induced response in human islet cells by withaferin A.

PubMed

Peng, H; Olsen, G; Tamura, Y; Noguchi, H; Matsumoto, S; Levy, M F; Naziruddin, B

2010-01-01

After islet cell transplantation, a substantial mass of islets are lost owing to nonspecific inflammatory reactions. Cytokine exposure before or after transplantation can upregulate expression of proinflammatory genes via the nuclear factor-kappaB signaling pathway, eventually resulting in islet loss. To test the effects of a naturally occurring nuclear factor-kappaB inhibitor, withaferin A, on regulation of inflammatory genes in human islets. Human pancreatic islets were isolated using a modified Ricordi protocol. Purified islets were cultured for 2 days. The effect of withaferin A treatment on islet cell viability was examined using the fluorescein diacetate-propidium iodide dye exclusion test, and on function using a static glucose stimulation assay. Islet cells were treated with a cytokine mixture (50 U/mL of interleukin-1beta, 1000 U/mL of tumor necrosis factor-alpha, and 1000 U/mL of interferon-gamma) for 48 hours with or without withaferin A, 1 microg/mL. Treated islets were used for real-time polymerase chain reaction (PCR) array analysis for expression of inflammatory genes, and expression of other selected genes was analyzed using real-time PCR with single primers. Glucose stimulation and viability assays demonstrated that withaferin A was not toxic to islet cells. Of 84 inflammation-related genes examined using real-time PCR array analysis, 9 were significantly upregulated by cytokine treatment compared with the control group. However, addition of withaferin A to the culture significantly inhibited expression of all genes. Withaferin A significantly inhibits the inflammatory response of islet cells with cytokine exposure. Copyright 2010 Elsevier Inc. All rights reserved.

A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

PubMed

Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li

2017-01-01

Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P < 0.05). There was no significant difference in islet viability among the four groups. The islet purity, function, yield, and cost of our method were superior to those of the Ficoll-400 and 1077 methods, but inferior to the handpicking method. However, the handpicking method may cause wrist injury and visual impairment in researchers during large-scale islet isolation (>1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

NASA Astrophysics Data System (ADS)

Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

1982-08-01

Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

A novel method for murine intrahepatic islet transplantation via cecal vein.

PubMed

Byun, Nari; Kim, Hyun-Je; Min, Byoung-Hoon; Shin, Jun-Seop; Yoon, Il-Hee; Kim, Jong-Min; Kim, Yong-Hee; Park, Chung-Gyu

2015-12-01

Islet transplantation is one of the most beneficial treatment modality to treat type 1 diabetic patients with frequent hypoglycemic unawareness. In clinical setting, human islets are infused via portal vein and are settled in the end-portal venules in the liver. However, mouse islets are transplanted into kidney subcapsule or liver through direct portal vein. These conventional transplantation methods have several drawbacks such as different physiological environments around the transplanted islets in kidney subcapsule from the liver and high mortality rate in direct portal vein approach. In this study, we introduced murine intrahepatic islet transplantation method via cecal vein to have the same surgical operation route in humans as well as guaranteeing low mortality rate after islet transplantation. With this protocol, consistent normoglycemia can be obtained in diabetic mice, while keeping operation-related mortality extremely low. This approach with easier accessibility and low mortality will make murine intrahepatic islet transplantation a useful model for studying immunological mechanisms such as strong innate and adaptive immune responses that occur in human islet transplantation. Copyright © 2015 Elsevier B.V. All rights reserved.

Transplantation of co-aggregates of Sertoli cells and islet cells into liver without immunosuppression.

PubMed

Takemoto, Naohiro; Liu, Xibao; Takii, Kento; Teramura, Yuji; Iwata, Hiroo

2014-02-15

Transplantation of islets of Langerhans (islets) was used to treat insulin-dependent diabetes mellitus. However, islet grafts must be maintained by administration of immunosuppressive drugs, which can lead to complications in the long term. An approach that avoids immunosuppressive drug use is desirable. Co-aggregates of Sertoli cells and islet cells from BALB/c mice that were prepared by the hanging drop method were transplanted into C57BL/6 mouse liver through the portal vein as in human clinical islet transplantation. The core part of the aggregates contained mainly Sertoli cells, and these cells were surrounded by islet cells. The co-aggregates retained the functions of both Sertoli and islet cells. When 800 co-aggregates were transplanted into seven C57BL/6 mice via the portal vein, six of seven recipient mice demonstrated quasi-normoglycemia for more than 100 days. The hanging drop method is suitable for preparing aggregates of Sertoli and islet cells for transplantation. Notably, transplantation of these allogeneic co-aggregates into mice with chemically induced diabetes via the portal vein resulted in long-term graft survival without systemic immunosuppression.

Enhanced colonic delivery of ciclosporin A self-emulsifying drug delivery system encapsulated in coated minispheres.

PubMed

Keohane, Kieran; Rosa, Mónica; Coulter, Ivan S; Griffin, Brendan T

2016-01-01

Investigate the potential of coated minispheres (SmPill®) to enhance localized Ciclosporin A (CsA) delivery to the colon. CsA self-emulsifying drug delivery systems (SEDDS) were encapsulated into SmPill® minispheres. Varying degrees of coating thickness (low, medium and high) were applied using ethylcellulose and pectin (E:P) polymers. In vitro CsA release was evaluated in simulated gastric and intestinal media. Bioavailability of CsA in vivo following oral administration to pigs of SmPill® minispheres was compared to Neoral® po and Sandimmun® iv in a pig model. CsA concentrations in blood and intestinal tissue were determined by HPLC-UV. In vitro CsA release from coated minispheres decreased with increasing coating thickness. A linear relationship was observed between in vitro CsA release and in vivo bioavailability (r(2) = 0.98). CsA concentrations in the proximal, transverse and distal colon were significantly higher following administration of SmPill®, compared to Neoral® po and Sandimmun® iv (p < 0.05). Analysis of transverse colon tissue subsections also revealed significantly higher CsA concentrations in the mucosa and submucosa using SmPill® minispheres (p < 0.05). Modulating E:P coating thickness controls release of CsA from SmPill® minispheres. Coated minispheres limited CsA release in the small intestine and enhanced delivery and uptake in the colon. These findings demonstrate clinical advantages of an oral coated minisphere-enabled CsA formulation in the treatment of inflammatory conditions of the large intestine.

Current issues in allogeneic islet transplantation.

PubMed

Chang, Charles A; Lawrence, Michael C; Naziruddin, Bashoo

2017-10-01

Transplantation of allogenic pancreatic islets is a minimally invasive treatment option to control severe hypoglycemia and dependence on exogenous insulin among type 1 diabetes (T1D) patients. This overview summarizes the current issues and progress in islet transplantation outcomes and research. Several clinical trials from North America and other countries have documented the safety and efficacy of clinical islet transplantation for T1D patients with impaired hypoglycemia awareness. A recently completed phase 3 clinical trial allows centres in the United States to apply for a Food and Drug Administration Biologics License for the procedure. Introduction of anti-inflammatory drugs along with T-cell depleting induction therapy has significantly improved long-term function of transplanted islets. Research into islet biomarkers, immunosuppression, extrahepatic transplant sites and potential alternative beta cell sources is driving further progress. Allogeneic islet transplantation has vastly improved over the past two decades. Success in restoration of glycemic control and hypoglycemic awareness after islet transplantation has been further highlighted by clinical trials. However, lack of effective strategies to maintain long-term islet function and insufficient sources of donor tissue still impose limitations to the widespread use of islet transplantation. In the United States, wide adoption of this technology still awaits regulatory approval and, importantly, a financial mechanism to support the use of this technology.

Islet oxygen consumption rate (OCR) dose predicts insulin independence for first clinical islet allotransplants

PubMed Central

Kitzmann, JP; O’Gorman, D; Kin, T; Gruessner, AC; Senior, P; Imes, S; Gruessner, RW; Shapiro, AMJ; Papas, KK

2014-01-01

Human islet allotransplant (ITx) for the treatment of type 1 diabetes is in phase III clinical registration trials in the US and standard of care in several other countries. Current islet product release criteria include viability based on cell membrane integrity stains, glucose stimulated insulin release (GSIR), and islet equivalent (IE) dose based on counts. However, only a fraction of patients transplanted with islets that meet or exceed these release criteria become insulin independent following one transplant. Measurements of islet oxygen consumption rate (OCR) have been reported as highly predictive of transplant outcome in many models. In this paper we report on the assessment of clinical islet allograft preparations using islet oxygen consumption rate (OCR) dose (or viable IE dose) and current product release assays in a series of 13 first transplant recipients. The predictive capability of each assay was examined and successful graft function was defined as 100% insulin independence within 45 days post-transplant. Results showed that OCR dose was most predictive of CTO. IE dose was also highly predictive, while GSIR and membrane integrity stains were not. In conclusion, OCR dose can predict CTO with high specificity and sensitivity and is a useful tool for evaluating islet preparations prior to clinical ITx. PMID:25131089

Impact of Pancreatic Rat Islet Density on Cell Survival during Hypoxia

PubMed Central

Rodriguez-Brotons, A.; Bietiger, W.; Peronet, C.; Magisson, J.; Sookhareea, C.; Langlois, A.; Mura, C.; Jeandidier, N.; Pinget, M.; Sigrist, S.; Maillard, E.

2016-01-01

In bioartificial pancreases (BP), the number of islets needed to restore normoglycaemia in the diabetic patient is critical. However, the confinement of a high quantity of islets in a limited space may impact islet survival, particularly in regard to the low oxygen partial pressure (PO2) in such environments. The aim of the present study was to evaluate the impact of islet number in a confined space under hypoxia on cell survival. Rat islets were seeded at three different concentrations (150, 300, and 600 Islet Equivalents (IEQ)/cm2) and cultured in normal atmospheric pressure (160 mmHg) as well as hypoxic conditions (15 mmHg) for 24 hours. Cell viability, function, hypoxia-induced changes in gene expression, and cytokine secretion were then assessed. Notably, hypoxia appeared to induce a decrease in viability and increasing islet density exacerbated the observed increase in cellular apoptosis as well as the loss of function. These changes were also associated with an increase in inflammatory gene transcription. Taken together, these data indicate that when a high number of islets are confined to a small space under hypoxia, cell viability and function are significantly impacted. Thus, in order to improve islet survival in this environment during transplantation, oxygenation is of critical importance. PMID:26824040

The lizard fauna of Guam's fringing islets: Island biogeography, phylogenetic history, and conservation implications

USGS Publications Warehouse

Perry, G.; Rodda, G.H.; Fritts, T.H.; Sharp, T.R.

1998-01-01

We sampled the lizard fauna of twenty-two small islets fringing the Pacific island of Guam and used these data to shed light on the processes responsible for present-day diversity. Habitat diversity, measured by islet area and vegetation complexity, was significantly correlated with the number of species found on an islet. However, islet distance and elevation were not significant predictors of diversity. Distribution patterns were slightly different for the two major families in our sample, Scincidae and Gekkonidae: skinks needed larger islets to maintain a population than did geckos. Presence/absence patterns were highly and significantly nested, and population density was correlated with the number of islets on which a species was found. An area cladogram was poorly supported and showed no faunal similarity between nearby islands. These patterns indicate that extinctions on most islets were due mostly to non-catastrophic, long-acting biological causes. The presence on the islets of species extirpated on Guam and the lack of significant nestedness on islands with greater maximum elevation highlight the impact that predators (primarily brown treesnakes) can have. Our findings also show that small reserves will not suffice to protect endangered lizard faunas, and that the islets may serve as a short-term repository of such species until snake-free areas can be established on Guam.

Modular tissue engineering for the vascularization of subcutaneously transplanted pancreatic islets

PubMed Central

Vlahos, Alexander E.; Cober, Nicholas; Sefton, Michael V.

2017-01-01

The transplantation of pancreatic islets, following the Edmonton Protocol, is a promising treatment for type I diabetics. However, the need for multiple donors to achieve insulin independence reflects the large loss of islets that occurs when islets are infused into the portal vein. Finding a less hostile transplantation site that is both minimally invasive and able to support a large transplant volume is necessary to advance this approach. Although the s.c. site satisfies both these criteria, the site is poorly vascularized, precluding its utility. To address this problem, we demonstrate that modular tissue engineering results in an s.c. vascularized bed that enables the transplantation of pancreatic islets. In streptozotocin-induced diabetic SCID/beige mice, the injection of 750 rat islet equivalents embedded in endothelialized collagen modules was sufficient to restore and maintain normoglycemia for 21 days; the same number of free islets was unable to affect glucose levels. Furthermore, using CLARITY, we showed that embedded islets became revascularized and integrated with the host’s vasculature, a feature not seen in other s.c. studies. Collagen-embedded islets drove a small (albeit not significant) shift toward a proangiogenic CD206+MHCII−(M2-like) macrophage response, which was a feature of module-associated vascularization. While these results open the potential for using s.c. islet delivery as a treatment option for type I diabetes, the more immediate benefit may be for the exploration of revascularized islet biology. PMID:28814629

Pancreatic β-Cell-Derived IP-10/CXCL10 Isletokine Mediates Early Loss of Graft Function in Islet Cell Transplantation.

PubMed

Yoshimatsu, Gumpei; Kunnathodi, Faisal; Saravanan, Prathab Balaji; Shahbazov, Rauf; Chang, Charles; Darden, Carly M; Zurawski, Sandra; Boyuk, Gulbahar; Kanak, Mazhar A; Levy, Marlon F; Naziruddin, Bashoo; Lawrence, Michael C

2017-11-01

Pancreatic islets produce and secrete cytokines and chemokines in response to inflammatory and metabolic stress. The physiological role of these "isletokines" in health and disease is largely unknown. We observed that islets release multiple inflammatory mediators in patients undergoing islet transplants within hours of infusion. The proinflammatory cytokine interferon-γ-induced protein 10 (IP-10/CXCL10) was among the highest released, and high levels correlated with poor islet transplant outcomes. Transgenic mouse studies confirmed that donor islet-specific expression of IP-10 contributed to islet inflammation and loss of β-cell function in islet grafts. The effects of islet-derived IP-10 could be blocked by treatment of donor islets and recipient mice with anti-IP-10 neutralizing monoclonal antibody. In vitro studies showed induction of the IP-10 gene was mediated by calcineurin-dependent NFAT signaling in pancreatic β-cells in response to oxidative or inflammatory stress. Sustained association of NFAT and p300 histone acetyltransferase with the IP-10 gene required p38 and c-Jun N-terminal kinase mitogen-activated protein kinase (MAPK) activity, which differentially regulated IP-10 expression and subsequent protein release. Overall, these findings elucidate an NFAT-MAPK signaling paradigm for induction of isletokine expression in β-cells and reveal IP-10 as a primary therapeutic target to prevent β-cell-induced inflammatory loss of graft function after islet cell transplantation. © 2017 by the American Diabetes Association.

Diabetes Is Reversed in a Murine Model by Marginal Mass Syngeneic Islet Transplantation Using a Subcutaneous Cell Pouch Device

PubMed Central

Pepper, Andrew R.; Pawlick, Rena; Gala-Lopez, Boris; MacGillivary, Amanda; Mazzuca, Delfina M.; White, David J. G.; Toleikis, Philip M.; Shapiro, A. M. James

2015-01-01

Background Islet transplantation is a successful β-cell replacement therapy for selected patients with type 1 diabetes mellitus. Although high rates of early insulin independence are achieved routinely, long-term function wanes over time. Intraportal transplantation is associated with procedural risks, requires multiple donors, and does not afford routine biopsy. Stem cell technologies may require potential for retrievability, and graft removal by hepatectomy is impractical. There is a clear clinical need for an alternative, optimized transplantation site. The subcutaneous space is a potential substitute, but transplantation of islets into this site has routinely failed to reverse diabetes. However, an implanted device, which becomes prevascularized before transplantation, may alter this equation. Methods Syngeneic mouse islets were transplanted subcutaneously within Sernova Corp's Cell Pouch (CP). All recipients were preimplanted with CPs 4 weeks before diabetes induction and transplantation. After transplantation, recipients were monitored for glycemic control and glucose tolerance. Results Mouse islets transplanted into the CP routinely restored glycemic control with modest delay and responded well to glucose challenge, comparable to renal subcapsular islet grafts, despite a marginal islet dose, and normoglycemia was maintained until graft explantation. In contrast, islets transplanted subcutaneously alone failed to engraft. Islets within CPs stained positively for insulin, glucagon, and microvessels. Conclusions The CP is biocompatible, forms an environment suitable for islet engraftment, and offers a potential alternative to the intraportal site for islet and future stem cell therapies. PMID:26308506

Comparison of exendin-4 on beta-cell replication in mouse and human islet grafts.

PubMed

Tian, Lei; Gao, Jie; Weng, Guangbin; Yi, Huimin; Tian, Bole; O'Brien, Timothy D; Guo, Zhiguang

2011-08-01

Exendin-4 can stimulate β-cell replication in mice. Whether it can stimulate β-cell replication in human islet grafts remains unknown. Therefore, we compared the effects of exendin-4 on β-cell replication in mouse and human islet grafts. Islets, isolated from mouse and human donors at different ages, were transplanted into diabetic mice and/or diabetic nude mice that were given bromodeoxyuridine (BrdU) with or without exendin-4. At 4 weeks post-transplantation, islet grafts were removed for insulin and BrdU staining and quantification of insulin(+)/BrdU(+) cells. Although diabetes was reversed in all mice transplanting syngeneic mouse islets from young or old donors, normoglycemia was achieved significantly faster in exendin-4 treated mice. Mouse islet grafts in exendin-4 treated mice had significantly more insulin(+)/BrdU(+) β cells than in untreated mice (P < 0.01). Human islet grafts from ≤22-year-old donors had more insulin(+)/BrdU(+) β cells in exendin-4 treated mice than that in untreated mice (P < 0.01). However, human islet grafts from ≥35-year-old donors contained few insulin(+)/BrdU(+) β cells in exendin-4 treated or untreated mice. Our data demonstrated that the capacity for β-cell replication in mouse and human islet grafts is different with and without exendin-4 treatment and indicated that GLP-1 agonists can stimulate β-cell replication in human islets from young donors. © 2011 The Authors. Transplant International © 2011 European Society for Organ Transplantation.

Functional imaging of glucose-evoked rat islet activities using transient intrinsic optical signals

NASA Astrophysics Data System (ADS)

Yao, Xin-Cheng; Cui, Wan-Xing; Li, Yi-Chao; Zhang, Wei; Lu, Rong-Wen; Thompson, Anthony; Amthor, Franklin; Wang, Xu-Jing

2012-05-01

We demonstrate intrinsic optical signal (IOS) imaging of intact rat islet, which consists of many endocrine cells working together. A near-infrared digital microscope was employed for optical monitoring of islet activities evoked by glucose stimulation. Dynamic NIR images revealed transient IOS responses in the islet activated by low-dose (2.75 mM) and high-dose (5.5 mM) glucose stimuli. Comparative experiments and quantitative analysis indicated that both glucose metabolism and calcium/insulin dynamics might contribute to the observed IOS responses. Further investigation of the IOS imaging technology may provide a high resolution method for ex vivo functional examination of the islet, which is important for advanced study of diabetes associated islet dysfunctions and for improved quality control of donor islets for transplantation.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets

PubMed Central

KIM, JAEHYUP; BREUNIG, MELISSA J.; ESCALANTE, LEAH E.; BHATIA, NEEHAR; DENU, RYAN A.; DOLLAR, BRIDGET A.; STEIN, ANDREW P.; HANSON, SUMMER E.; NADERI, NADIA; RADEK, JAMES; HAUGHY, DERMOT; BLOOM, DEBRA D.; ASSADI-PORTER, FARIBA M.; HEMATTI, PEIMAN

2012-01-01

Background aims Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. Methods We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. Results Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. Conclusions Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts. PMID:22571381

Potential differentiation of islet-like cells from pregnant cow-derived placental stem cells.

PubMed

Peng, Shao-Yu; Chou, Chien-Wen; Kuo, Yu-Hsuan; Shen, Perng-Chih; Shaw, S W Steven

2017-06-01

Type 1 diabetes is an autoimmune disease that destroys islet cells and results in insufficient insulin secretion by pancreatic β-cells. Islet transplantation from donors is an approach used for treating patients with diabetes; however, this therapy is difficult to implement because of the lack of donors. Nevertheless, several stem cells have the potential to differentiate from islet-like cells and enable insulin secretion for treating diabetes in animal models. For example, placenta is considered a waste material and can be harvested noninvasively during delivery without ethical or moral concerns. To date, the differentiation of islet-like cells from cow-derived placental stem cells (CPSCs) has yet to be demonstrated. The investigation of potential differentiation of islet-like cells from CPSCs was conducted by supplementation with nicotinamide, exendin-4, glucose, and poly-d-lysine and was detected through reverse transcription polymerase chain reaction, dithizone staining, and immunocytochemical methods. Our results indicated that CPSCs are established and express mesenchymal stem cell surface antigen markers, such as CD73, CD166, β-integrin, and Oct-4, but not hematopoietic stem cell surface antigen markers, such as CD45. After induction, the CPSCs successfully differentiated into islet-like cells. The CPSC-derived islet-like cells expressed islet cell development-related genes, such as insulin, glucagon, pax-4, Nkx6.1, pax-6, and Fox. Moreover, CPSC-derived islet-like cells can be stained with zinc ions, which are widely distributed in the islet cells and enable insulin secretion. Altogether, islet-like cells have the potential to be differentiated from CPSCs without gene manipulation, and can be used in diabetic animal models in the future for preclinical and drug testing trial investigations. Copyright © 2017. Published by Elsevier B.V.

Importance of extranuclear estrogen receptor-alpha and membrane G protein-coupled estrogen receptor in pancreatic islet survival.

PubMed

Liu, Suhuan; Le May, Cedric; Wong, Winifred P S; Ward, Robert D; Clegg, Deborah J; Marcelli, Marco; Korach, Kenneth S; Mauvais-Jarvis, Franck

2009-10-01

We showed that 17beta-estradiol (E(2)) favors pancreatic beta-cell survival via the estrogen receptor-alpha (ERalpha) in mice. E(2) activates nuclear estrogen receptors via an estrogen response element (ERE). E(2) also activates nongenomic signals via an extranuclear form of ERalpha and the G protein-coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival. We used mice and islets deficient in estrogen receptor-alpha (alphaERKO(-/-)), estrogen receptor-beta (betaERKO(-/-)), estrogen receptor-alpha and estrogen receptor-beta (alphabetaERKO(-/-)), and GPER (GPERKO(-/-)); a mouse lacking ERalpha binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes. We show that ERalpha protection of islet survival is ERE independent and that E(2) favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERbeta plays a minor cytoprotective role compared to ERalpha. Accordingly, betaERKO(-/-) mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERalpha and ERbeta in mice does not synergize to provoke islet apoptosis. In alphabetaERKO(-/-) mice and their islets, E(2) partially prevents apoptosis suggesting that an alternative pathway compensates for ERalpha/ERbeta deficiency. We find that E(2) protection of islet survival is reproduced by a membrane-impermeant E(2) formulation and a selective GPER agonist. Accordingly, GPERKO(-/-) mice are susceptible to streptozotocin-induced insulin deficiency. E(2) protects beta-cell survival through ERalpha and ERbeta via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in beta-cells and identifies GPER as a target to protect islet survival.

Insulinotropic and antidiabetic effects of 17β-estradiol and the GPR30 agonist G-1 on human pancreatic islets.

PubMed

Kumar, Rajesh; Balhuizen, Alexander; Amisten, Stefan; Lundquist, Ingmar; Salehi, Albert

2011-07-01

We have recently shown that 17β-estradiol (E2) and the synthetic G protein-coupled receptor 30 (GPR30) ligand G-1 have antiapoptotic actions in mouse pancreatic islets, raising the prospect that they might exert beneficial effects also in human islets. The objective of the present study was to identify the expression of GPR30 in human islets and clarify the role of GPR30 in islet hormone secretion and β-cell survival. GPR30 expression was analyzed by confocal microscopy, Western blot, and quantitative PCR in islets from female and male donors. Hormone secretion, phosphatidylinositol hydrolysis, cAMP content, and caspase-3 activity in female islets were determined with conventional methods and apoptosis with the annexin-V method. Confocal microscopy revealed GPR30 expression in islet insulin, glucagon, and somatostatin cells. GPR30 mRNA and protein expression was markedly higher in female vs. male islets. An amplifying effect of G-1 or E2 on cAMP content and insulin secretion from isolated female islets was not influenced by the E2 genomic receptor (ERα and ERβ) antagonists ICI 182,780 and EM-652. Cytokine-induced (IL-1β plus TNFα plus interferon-γ) apoptosis in islets cultured for 24 h at 5 mmol/liter glucose was almost abolished by G-1 or E2 treatment and was not affected by the nuclear estrogen receptor antagonists. Concentration-response studies on female islets from healthy controls and type 2 diabetic subjects showed that both E2 and G-1 displayed important antidiabetic actions by improving glucose-stimulated insulin release while suppressing glucagon and somatostatin secretion. In view of these findings, we propose that small molecules activating GPR30 could be promising in the therapy of diabetes mellitus.

Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

PubMed

Papas, Klearchos K; Bellin, Melena D; Sutherland, David E R; Suszynski, Thomas M; Kitzmann, Jennifer P; Avgoustiniatos, Efstathios S; Gruessner, Angelika C; Mueller, Kathryn R; Beilman, Gregory J; Balamurugan, Appakalai N; Loganathan, Gopalakrishnan; Colton, Clark K; Koulmanda, Maria; Weir, Gordon C; Wilhelm, Josh J; Qian, Dajun; Niland, Joyce C; Hering, Bernhard J

2015-01-01

Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR) in predicting clinical islet autotransplant (IAT) insulin independence (II). IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity. Membrane integrity staining (FDA/PI), OCR normalized to DNA (OCR/DNA), islet equivalent (IE) and OCR (viable IE) normalized to recipient body weight (IE dose and OCR dose), and OCR/DNA normalized to islet size index (ISI) were used to characterize autoislet preparations (n = 35). Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis. Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001). These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC) = 0.94 for IE dose and 0.96 for OCR dose). FDA/PI (AUC = 0.49) and OCR/DNA (AUC = 0.58) did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72). Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

Local Expression of Indoleamine 2,3 Dioxygenase in Syngeneic Fibroblasts Significantly Prolongs Survival of an Engineered Three-Dimensional Islet Allograft

PubMed Central

Jalili, Reza B.; Forouzandeh, Farshad; Rezakhanlou, Alireza Moeen; Hartwell, Ryan; Medina, Abelardo; Warnock, Garth L.; Larijani, Bagher; Ghahary, Aziz

2010-01-01

OBJECTIVE The requirement of systemic immunosuppression after islet transplantation is of significant concern and a major drawback to clinical islet transplantation. Here, we introduce a novel composite three-dimensional islet graft equipped with a local immunosuppressive system that prevents islet allograft rejection without systemic antirejection agents. In this composite graft, expression of indoleamine 2,3 dioxygenase (IDO), a tryptophan-degrading enzyme, in syngeneic fibroblasts provides a low-tryptophan microenvironment within which T-cells cannot proliferate and infiltrate islets. RESEARCH DESIGN AND METHODS Composite three-dimensional islet grafts were engineered by embedding allogeneic mouse islets and adenoviral-transduced IDO–expressing syngeneic fibroblasts within collagen gel matrix. These grafts were then transplanted into renal subcapsular space of streptozotocin diabetic immunocompetent mice. The viability, function, and criteria for graft take were then determined in the graft recipient mice. RESULTS IDO-expressing grafts survived significantly longer than controls (41.2 ± 1.64 vs. 12.9 ± 0.73 days; P < 0.001) without administration of systemic immunesuppressive agents. Local expression of IDO suppressed effector T-cells at the graft site, induced a Th2 immune response shift, generated an anti-inflammatory cytokine profile, delayed alloantibody production, and increased number of regulatory T-cells in draining lymph nodes, which resulted in antigen-specific impairment of T-cell priming. CONCLUSIONS Local IDO expression prevents cellular and humoral alloimmune responses against islets and significantly prolongs islet allograft survival without systemic antirejection treatments. This promising finding proves the potent local immunosuppressive activity of IDO in islet allografts and sets the stage for development of a long-lasting nonrejectable islet allograft using stable IDO induction in bystander fibroblasts. PMID:20522587

Isles within islets: The lattice origin of small-world networks in pancreatic tissues

NASA Astrophysics Data System (ADS)

Barua, Amlan K.; Goel, Pranay

2016-02-01

The traditional computational model of the pancreatic islets of Langerhans is a lattice of β-cells connected with gap junctions. Numerous studies have investigated the behavior of networks of coupled β-cells and have shown that gap junctions synchronize bursting strongly. This simplistic architecture of islets, however, seems increasingly untenable at the face of recent experimental advances. In a microfluidics experiment on isolated islets, Rocheleau et al. (2004) showed a failure of penetration of excitation when one end received high glucose and other end was not excited sufficiently; this suggested that gap junctions may not be efficient at inducing synchrony throughout the islet. Recently, Stozer et al. (2013) have argued that the functional networks of β-cells in an islet are small world. Their results implicate the existence of a few long-range connections among cells in the network. The physiological reason underlying this claim is not well understood. These studies cast doubt on the original lattice model that largely predict an all-or-none synchrony among the cells. Here we have attempted to reconcile these observations in a unified framework. We assume that cells in the islet are coupled randomly to their nearest neighbors with some probability, p. We simulated detailed β-cell bursting in such islets. By varying p systematically we were led to network parameters similar to those obtained by Stozer et al. (2013). We find that the networks within islets break up into components giving rise to smaller isles within the super structure-isles-within-islets, as it were. This structure can also account for the partial excitation seen by Rocheleau et al. (2004). Our updated view of islet architecture thus explains the paradox how islets can have strongly synchronizing gap junctions, and be weakly coordinated at the same time.

Inotuzumab Ozogamicin Murine Analog–Mediated B-Cell Depletion Reduces Anti-islet Allo- and Autoimmune Responses

PubMed Central

Carvello, Michele; Petrelli, Alessandra; Vergani, Andrea; Lee, Kang Mi; Tezza, Sara; Chin, Melissa; Orsenigo, Elena; Staudacher, Carlo; Secchi, Antonio; Dunussi-Joannopoulos, Kyri; Sayegh, Mohamed H.; Markmann, James F.; Fiorina, Paolo

2012-01-01

B cells participate in the priming of the allo- and autoimmune responses, and their depletion can thus be advantageous for islet transplantation. Herein, we provide an extensive study of the effect of B-cell depletion in murine models of islet transplantation. Islet transplantation was performed in hyperglycemic B-cell–deficient(μMT) mice, in a purely alloimmune setting (BALB/c into hyperglycemic C57BL/6), in a purely autoimmune setting (NOD.SCID into hyperglycemic NOD), and in a mixed allo-/autoimmune setting (BALB/c into hyperglycemic NOD). Inotuzumab ozogamicin murine analog (anti-CD22 monoclonal antibody conjugated with calicheamicin [anti-CD22/cal]) efficiently depleted B cells in all three models of islet transplantation examined. Islet graft survival was significantly prolonged in B-cell–depleted mice compared with control groups in transplants of islets from BALB/c into C57BL/6 (mean survival time [MST]: 16.5 vs. 12.0 days; P = 0.004), from NOD.SCID into NOD (MST: 23.5 vs. 14.0 days; P = 0.03), and from BALB/c into NOD (MST: 12.0 vs. 5.5 days; P = 0.003). In the BALB/c into B-cell–deficient mice model, islet survival was prolonged as well (MST: μMT = 32.5 vs. WT = 14 days; P = 0.002). Pathology revealed reduced CD3+ cell islet infiltration and confirmed the absence of B cells in treated mice. Mechanistically, effector T cells were reduced in number, concomitant with a peripheral Th2 profile skewing and ex vivo recipient hyporesponsiveness toward donor-derived antigen as well as islet autoantigens. Finally, an anti-CD22/cal and CTLA4-Ig–based combination therapy displayed remarkable prolongation of graft survival in the stringent model of islet transplantation (BALB/c into NOD). Anti-CD22/cal–mediated B-cell depletion promotes the reduction of the anti-islet immune response in various models of islet transplantation. PMID:22076927

Improvement of porcine islet isolation by inhibition of trypsin activity during pancreas preservation and digestion using α1-antitrypsin.

PubMed

Shimoda, Masayuki; Noguchi, Hirofumi; Fujita, Yasutaka; Takita, Morihito; Ikemoto, Tetsuya; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F; Kobayashi, Naoya; Grayburn, Paul A; Matsumoto, Shinichi

2012-01-01

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether α1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Desnutrin/ATGL Activates PPARδ to Promote Mitochondrial Function for Insulin Secretion in Islet β cells

PubMed Central

Tang, Tianyi; Abbott, Marcia J.; Ahmadian, Maryam; Lopes, Andressa B.; Wang, Yuhui; Sul, Hei Sook

2013-01-01

Excessive caloric intake leading to obesity is associated with insulin resistance and dysfuntion of islet β cells. High fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (βKO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. βKO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPARδ target genes involved in mitochondrial oxidation. Furthermore, synthetic PPARδ, but not PPARα, agonist restores GSIS and expression of mitochondrial oxidative genes in βKO mice, revealing desnutrin-catalyzed lipolysis generates PPARδ ligands. Finally, adenoviral expression of desnutrin in βKO islets restores all defects of βKO islet phenotype and function including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPARδ-mitochondrial oxidation axis in regulating islet β cell GSIS. PMID:24268737

Glucose Oscillations Can Activate an Endogenous Oscillator in Pancreatic Islets

PubMed Central

Mukhitov, Nikita; Roper, Michael G.; Bertram, Richard

2016-01-01

Pancreatic islets manage elevations in blood glucose level by secreting insulin into the bloodstream in a pulsatile manner. Pulsatile insulin secretion is governed by islet oscillations such as bursting electrical activity and periodic Ca2+ entry in β-cells. In this report, we demonstrate that although islet oscillations are lost by fixing a glucose stimulus at a high concentration, they may be recovered by subsequently converting the glucose stimulus to a sinusoidal wave. We predict with mathematical modeling that the sinusoidal glucose signal’s ability to recover islet oscillations depends on its amplitude and period, and we confirm our predictions by conducting experiments with islets using a microfluidics platform. Our results suggest a mechanism whereby oscillatory blood glucose levels recruit non-oscillating islets to enhance pulsatile insulin output from the pancreas. Our results also provide support for the main hypothesis of the Dual Oscillator Model, that a glycolytic oscillator endogenous to islet β-cells drives pulsatile insulin secretion. PMID:27788129

Rapid deposition of amyloid in human islets transplanted into nude mice.

PubMed

Westermark, P; Eizirik, D L; Pipeleers, D G; Hellerström, C; Andersson, A

1995-05-01

Human islets of Langerhans were transplanted to the subcapsular space of the kidneys of nude mice which were either normoglycaemic or made diabetic with alloxan. After 2 weeks, the transplants were processed for light and electron microscopical analyses. In all transplants, islet amyloid polypeptide (IAPP)-positive cells were found with highest frequency in normoglycaemic animals. IAPP-positive amyloid was seen in 16 out of 22 transplants (73%), either by polarisation microscopy after Congo red staining or by immune electron microscopy. At variance with previous findings of amyloid deposits exclusively in the extracellular space of islets of non-insulin-dependent diabetic patients, the grafted islets contained intracellular amyloid deposits as well. There was no clear difference in occurrence of amyloid between diabetic and non-diabetic animals. The present study indicates that human islets transplanted into nude mice very soon present IAPP-positive amyloid deposits. This technique may provide a valuable model for studies of the pathogenesis of islet amyloid and its impact on islet cell function.

Microcirculation of human pancreatic islets transplanted under the renal capsule of nude mice.

PubMed

Jansson, L; Tyrberg, B; Carlsson, P O; Nordin, A; Andersson, A; Källskog O

2001-08-27

The aim was to measure the capillary blood pressure in transplanted human islets. Human islets were isolated at the Central Unit of the beta-cell Transplant in Brussels, Belgium. After transport to our laboratory, the islets were implanted under the renal capsule of normoglycemic nude mice. Two weeks later the capillary and venous blood pressures in the islet graft and adjacent renal parenchyma were measured with a micropuncture technique. Capillary blood pressure was approximately 5-8 mmHg in both graft and renal capillaries: twice as high as in native islets. Venous blood pressures were similar (4-5 mmHg) in the veins draining the graft and in the renal interlobular veins. All veins leading from the graft emptied into the renal parenchyma, that is, into interlobular veins. The capillary hypertension seen in transplanted human islets is probably necessary to secure adequate drainage through the renal veins. Whether this contributes to the poor results of long-term islet graft survival is unknown.

Assessment of six different collagenase-based methods to isolate feline pancreatic islets.

PubMed

Zini, Eric; Franchini, Marco; Guscetti, Franco; Osto, Melania; Kaufmann, Karin; Ackermann, Mathias; Lutz, Thomas A; Reusch, Claudia E

2009-12-01

Isolation of pancreatic islets is necessary to study the molecular mechanisms underlying beta-cell demise in diabetic cats. Six collagenase-based methods of isolation were compared in 10 cat pancreata, including single and double course of collagenase, followed or not by Ficoll centrifugation or accutase, and collagenase plus accutase. Morphometric analysis was performed to measure the relative area of islet and exocrine tissue. Islet specific mRNA transcripts were quantified in isolates by real-time PCR. The single and double course of collagenase digestion was successful in each cat and provided similar islet-to-exocrine tissue ratio. Quantities of insulin mRNA did not differ between the two methods. However, on histological examination either method yielded only approximately 2% of pure islets. The other methods provided disrupted islets or insufficient samples in 1-7 cats. Although pancreas digestion with single and double course of collagenase was superior, further studies are needed to improve islet isolation in cats.

Local immunomodulation with Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance.

PubMed

Headen, Devon M; Woodward, Kyle B; Coronel, María M; Shrestha, Pradeep; Weaver, Jessica D; Zhao, Hong; Tan, Min; Hunckler, Michael D; Bowen, William S; Johnson, Christopher T; Shea, Lonnie; Yolcu, Esma S; García, Andrés J; Shirwan, Haval

2018-06-04

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T effector cells are responsible for islet allograft rejection and express Fas death receptors following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of the Fas ligand with streptavidin (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4 + CD25 + FoxP3 + T regulatory cells in the graft and draining lymph nodes. Deletion of T regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

Application of Rotating Wall Vessel (RWV) Cell Culture for Pancreas Islet Cell Transplantation

NASA Technical Reports Server (NTRS)

Rutzky, Lynne P.

1998-01-01

Type I insulin-dependent diabetes mellitus (IDDM) remains a major cause of morbidity and mortality in both pediatric and adult populations, despite significant advances in medical management. While insulin therapy treats symptoms of acute diabetes, it fails to prevent chronic complications such as microvascular disease, blindness, neuropathy, and chronic renal failure. Strict control of blood glucose concentrations delays but does not prevent the onset and progression of secondary complications. Although, whole pancreas transplantation restores physiological blood glucose levels, a continuous process of allograft rejection causes vascular and exocrine-related complications. Recent advances in methods for isolation and purification of pancreatic islets make transplantation of islet allografts an attractive alternative to whole pancreas transplantation. However, immunosuppressive drugs are necessary to prevent rejection of islet allografts and many of these drugs are known to be toxic to the islets. Since auto-transplants of isolated islets following total pancreatectomy survive and function in vivo, it is apparent that a major obstacle to successful clinical islet transplantation is the immunogenicity of the islet allografts.

Streptozotocin Diabetes CORRELATION WITH EXTENT OF DEPRESSION OF PANCREATIC ISLET NICOTINAMIDE ADENINE DINUCLEOTIDE

PubMed Central

Anderson, Tom; Schein, Philip S.; McMenamin, Mary G.; Cooney, David A.

1974-01-01

The diabetogenic activity of streptozotocin has been correlated with a reduction in pyridine nucleotide synthesis in the mouse pancreatic islet. To determine the specificity of this reduction for diabetogenicity, a comparative study of streptozotocin, its cytotoxic moiety, 1-methyl-1-nitrosourea, and alloxan was performed. Streptozotocin administered intraperitoneally (i.p.) producd a dose-related reduction in islet NAD which was proportional to the degree of diabetogenicity. A diabetogenic dose, 200 mg/kg, attained a peak plasma N-nitroso intact streptozotocin concentration of 0.224 μmol/ml and reduced the mean islet NAD from a control of 0.78 to 0.15 pmol. At borderline, 150 mg/kg, and nondiabetogenic, 100 mg/kg, doses, plasma concentrations reached 0.161 and 0.136 μmol/ml, and NAD was 0.36 and 0.86 pmol/islet, respectively. 1-Methyl-1-nitrosourea, 100 mg/kg, attained a maximum N-nitroso intact 1-methyl-1-nitrosourea concentration of 0.162 μmol/ml and reduced the mean NAD to 0.58 pmol/islet, and was nondiabetogenic; 200 mg/kg attained a peak plasma concentration of 0.344 μmol/ml and depressed NAD to 0.38 pmol/islet, and was inconsistently diabetogenic. Islet NAD of 0.4 pmol/islet or greater is required for integrity of the beta cell. A diabetogenic dose of alloxan, 500 mg/kg, did not depress NAD, 0.85 pmol/islet, therefore confirming that its mechanism of diabetogenicity differs from that of streptozotocin. In vivo uptake of [methyl-14C]streptozotocin by islets was 3.8 times that of [methyl-14C]-1-methyl-1-nitrosourea, whereas uptake by the exocrine pancreas favored 1-methyl-1-nitrosourea over streptozotocin 2.4:1. The decreased islet uptake of 1-methyl-1-nitrosourea correlates with the 3.5 times increased molar dosage required to produce islet NAD depression comparable to that of streptozotocin, 150 mg/kg. These studies indicate that the glucose carrier of streptozotocin facilitates uptake of its cytotoxic group, 1-methyl-1-nitrosourea, into islets. PMID:4369217

Gap Junction Coupling and Calcium Waves in the Pancreatic Islet

PubMed Central

Benninger, Richard K. P.; Zhang, Min; Head, W. Steven; Satin, Leslie S.; Piston, David W.

2008-01-01

The pancreatic islet is a highly coupled, multicellular system that exhibits complex spatiotemporal electrical activity in response to elevated glucose levels. The emergent properties of islets, which differ from those arising in isolated islet cells, are believed to arise in part by gap junctional coupling, but the mechanisms through which this coupling occurs are poorly understood. To uncover these mechanisms, we have used both high-speed imaging and theoretical modeling of the electrical activity in pancreatic islets under a reduction in the gap junction mediated electrical coupling. Utilizing islets from a gap junction protein connexin 36 knockout mouse model together with chemical inhibitors, we can modulate the electrical coupling in the islet in a precise manner and quantify this modulation by electrophysiology measurements. We find that after a reduction in electrical coupling, calcium waves are slowed as well as disrupted, and the number of cells showing synchronous calcium oscillations is reduced. This behavior can be reproduced by computational modeling of a heterogeneous population of β-cells with heterogeneous levels of electrical coupling. The resulting quantitative agreement between the data and analytical models of islet connectivity, using only a single free parameter, reveals the mechanistic underpinnings of the multicellular behavior of the islet. PMID:18805925

Autologous Pancreatic Islet Transplantation in Human Bone Marrow

PubMed Central

Maffi, Paola; Balzano, Gianpaolo; Ponzoni, Maurilio; Nano, Rita; Sordi, Valeria; Melzi, Raffaella; Mercalli, Alessia; Scavini, Marina; Esposito, Antonio; Peccatori, Jacopo; Cantarelli, Elisa; Messina, Carlo; Bernardi, Massimo; Del Maschio, Alessandro; Staudacher, Carlo; Doglioni, Claudio; Ciceri, Fabio; Secchi, Antonio; Piemonti, Lorenzo

2013-01-01

The liver is the current site of choice for pancreatic islet transplantation, even though it is far from being ideal. We recently have shown in mice that the bone marrow (BM) may be a valid alternative to the liver, and here we report a pilot study to test feasibility and safety of BM as a site for islet transplantation in humans. Four patients who developed diabetes after total pancreatectomy were candidates for the autologous transplantation of pancreatic islet. Because the patients had contraindications for intraportal infusion, islets were infused in the BM. In all recipients, islets engrafted successfully as shown by measurable posttransplantation C-peptide levels and histopathological evidence of insulin-producing cells or molecular markers of endocrine tissue in BM biopsy samples analyzed during follow-up. Thus far, we have recorded no adverse events related to the infusion procedure or the presence of islets in the BM. Islet function was sustained for the maximum follow-up of 944 days. The encouraging results of this pilot study provide new perspectives in identifying alternative sites for islet infusion in patients with type 1 diabetes. Moreover, this is the first unequivocal example of successful engraftment of endocrine tissue in the BM in humans. PMID:23733196

Islet Assessment for Transplantation

PubMed Central

Papas, Klearchos K.; Suszynski, Thomas M.; Colton, Clark. K.

2010-01-01

Purpose of review There is a critical need for meaningful viability and potency assays that characterize islet preparations for release prior to clinical islet cell transplantation (ICT). Development, testing, and validation of such assays have been the subject of intense investigation for the past decade. These efforts are reviewed, highlighting the most recent results while focusing on the most promising assays. Recent Findings Assays based on membrane integrity do not reflect true viability when applied to either intact islets or dispersed islet cells. Assays requiring disaggregation of intact islets into individual cells for assessment introduce additional problems of cell damage and loss. Assays evaluating mitochondrial function, specifically mitochondrial membrane potential, bioenergetic status, and cellular oxygen consumption rate (OCR), especially when conducted with intact islets, appear most promising in evaluating their quality prior to ICT. Prospective, quantitative assays based on measurements of OCR with intact islets have been developed, validated and their results correlated with transplant outcomes in the diabetic nude mouse bioassay. Conclusion More sensitive and reliable islet viability and potency tests have been recently developed and tested. Those evaluating mitochondrial function are most promising, correlate with transplant outcomes in mice, and are currently being evaluated in the clinical setting. PMID:19812494

Non-Invasive Multiphoton Imaging of Islets Transplanted Into the Pinna of the NOD Mouse Ear Reveals the Immediate Effect of Anti-CD3 Treatment in Autoimmune Diabetes.

PubMed

Benson, Robert A; Garcon, Fabien; Recino, Asha; Ferdinand, John R; Clatworthy, Menna R; Waldmann, Herman; Brewer, James M; Okkenhaug, Klaus; Cooke, Anne; Garside, Paul; Wållberg, Maja

2018-01-01

We present a novel and readily accessible method facilitating cellular time-resolved imaging of transplanted pancreatic islets. Grafting of islets to the mouse ear pinna allows non-invasive, in vivo longitudinal imaging of events in the islets and enables improved acquisition of experimental data and use of fewer experimental animals than is possible using invasive techniques, as the same mouse can be assessed for the presence of islet infiltrating cells before and after immune intervention. We have applied this method to investigating therapeutic protection of beta cells through the well-established use of anti-CD3 injection, and have acquired unprecedented data on the nature and rapidity of the effect on the islet infiltrating T cells. We demonstrate that infusion of anti-CD3 antibody leads to immediate effects on islet infiltrating T cells in islet grafts in the pinna of the ear, and causes them to increase their speed and displacement within 20 min of infusion. This technique overcomes several technical challenges associated with intravital imaging of pancreatic immune responses and facilitates routine study of beta islet cell development, differentiation, and function in health and disease.

Portal Venous Oxygen Persufflation of the Donation after Cardiac Death pancreas in a rat model is superior to static cold storage and hypothermic machine perfusion.

PubMed

Reddy, Mettu S; Carter, Noel; Cunningham, Anne; Shaw, James; Talbot, David

2014-06-01

Success of clinical pancreatic islet transplantation depends on the mass of viable islets transplanted and the proportion of transplanted islets that survive early ischaemia reperfusion injury. Novel pancreas preservation techniques to improve islet preservation and viability can increase the utilization of donation after cardiac death donor pancreases for islet transplantation. Rat pancreases were retrieved after 30 min of warm ischaemia and preserved by static cold storage, hypothermic machine perfusion or retrograde portal venous oxygen persufflation for 6 h. They underwent collagenase digestion and density gradient separation to isolate islets. The yield, viability, morphology were compared. In vitro function of isolated islets was compared using glucose stimulated insulin secretion test. Portal venous oxygen persufflation improved the islet yield, viability and morphology as compared to static cold storage. The percentage of pancreases with good in vitro function (stimulation index > 1.0) was also higher after oxygen persufflation as compared to static cold storage. Retrograde portal venous oxygen persufflation of donation after cardiac death donor rat pancreases has the potential to improve islet yield. © 2014 Steunstichting ESOT.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wolf, B.A.; Easom, R.A.; Hughes, J.H.

Diacylglycerol accumulation has been examined in secretagogue-stimulated pancreatic islets with a newly developed negative ion chemical ionization mass spectrometric method. The muscarinic agonist carbachol induces islet accumulation of diacylglycerol rich in arachidonate and stearate, and a parallel accumulation of {sup 3}H-labeled diacylglycerol occurs in carbachol-stimulated islets that had been prelabeled with ({sup 3}H)glycerol. Islets so labeled do not accumulate {sup 3}H-labeled diacylglycerol in response to D-glucose, but D-glucose does induce islet accumulation of diacylglycerol by mass. This material is rich in palmitate and oleate and contains much smaller amounts of arachidonate. Neither secretagogue influences triacylglycerol labeling, and neither induces releasemore » of ({sup 3}H)choline or ({sup 3}H)phosphocholine from islets prelabeled with ({sup 3}H)choline. These observations indicate that the diacylglycerol that accumulates in islets in response to carbachol arises from hydrolysis of glycerolipids, probably including phosphoinositides. The bulk of the diacylglycerol which accumulates in response to glucose does not arise from glycerolipid hydrolysis and must therefore reflect de novo synthesis. The endogenous diacylglycerol which accumulates in secretagogue-stimulated islets may participate in insulin secretion because exogenous diacylglycerol induces insulin secretion from islets, and an inhibitor of diacylglycerol metabolism to phosphatidic acid augments glucose-induced insulin secretion.« less

Inhibition of Gelatinase B (Matrix Metalloprotease-9) Activity Reduces Cellular Inflammation and Restores Function of Transplanted Pancreatic Islets

PubMed Central

Lingwal, Neelam; Padmasekar, Manju; Samikannu, Balaji; Bretzel, Reinhard G.; Preissner, Klaus T.; Linn, Thomas

2012-01-01

Islet transplantation provides an approach to compensate for loss of insulin-producing cells in patients with type 1 diabetes. However, the intraportal route of transplantation is associated with instant inflammatory reactions to the graft and subsequent islet destruction as well. Although matrix metalloprotease (MMP)-2 and -9 are involved in both remodeling of extracellular matrix and leukocyte migration, their influence on the outcome of islet transplantation has not been characterized. We observed comparable MMP-2 mRNA expressions in control and transplanted groups of mice, whereas MMP-9 mRNA and protein expression levels increased after islet transplantation. Immunostaining for CD11b (Mac-1)-expressing leukocytes (macrophage, neutrophils) and Ly6G (neutrophils) revealed substantially reduced inflammatory cell migration into islet-transplanted liver in MMP-9 knockout recipients. Moreover, gelatinase inhibition resulted in a significant increase in the insulin content of transplanted pancreatic islets and reduced macrophage and neutrophil influx compared with the control group. These results indicate that the increase of MMP-9 expression and activity after islet transplantation is directly related to enhanced leukocyte migration and that early islet graft survival can be improved by inhibiting MMP-9 (gelatinase B) activity. PMID:22586582

Long-Term Follow-Up of the Edmonton Protocol of Islet Transplantation in the United States.

PubMed

Brennan, D C; Kopetskie, H A; Sayre, P H; Alejandro, R; Cagliero, E; Shapiro, A M J; Goldstein, J S; DesMarais, M R; Booher, S; Bianchine, P J

2016-02-01

We report the long-term follow-up of the efficacy and safety of islet transplantation in seven type 1 diabetic subjects from the United States enrolled in the multicenter international Edmonton Protocol who had persistent islet function after completion of the Edmonton Protocol. Subjects were followed up to 12 years with serial testing for sustained islet allograft function as measured by C-peptide. All seven subjects demonstrated continued islet function longer than a decade from the time of first islet transplantation. One subject remained insulin independent without the need for diabetic medications or supplemental transplants. One subject who was insulin-independent for over 8 years experienced graft failure 10.9 years after the first islet transplant. The remaining six subjects demonstrated continued islet function upon trial completion, although three had received a supplemental islet transplant each. At trial completion, five subjects were receiving insulin and two remained insulin independent, although one was treated with liraglutide. The median hemoglobin A1c was 6.3% (45 mmol/mol). All subjects experienced progressive decline in the C-peptide/glucose ratio. No patients experienced severe hypoglycemia, opportunistic infection, or lymphoma. Thus, although the rate and duration of insulin independence was low, the Edmonton Protocol was safe in the long term. Alternative approaches to islet transplantation are under investigation. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Current principles and practice in autologous intraportal islet transplantation: a meta-analysis of the technical considerations.

PubMed

Kumar, Rohan; Chung, Wen Yuan; Dennison, Ashley Robert; Garcea, Giuseppe

2016-04-01

Autologous islet transplantation (IAT) following pancreatectomy is now a recognized, albeit highly specialized procedure carried out in a small number of centers worldwide. Current clinical principles and best practice with emphasis on examining the technical aspects of surgery in centers with significant IAT experience are reviewed. Literature search for studies discussing any technical aspect of pancreatectomy with intraportal IAT was included. Thirty-five papers were included; all were single-center case series. The indications, surgical approach to pancreatectomy with IAT, islet yield, static pancreas preservation prior to islet digestion, portal vein access, absolute islet infusion volumes, and portal venous pressure changes during transfusion evaluated. IAT is considered a "last resort" when alternative approaches have been exhausted. Pre-morbid histology and prior surgical drainage adversely influence islet yields and may influence the clinical decision to perform pancreatectomy and IAT. Following pancreas digestion, absolute numbers of islets recovered and smaller islet size predict rates of insulin independence following IAT. Islet volumes and portal venous pressure changes are important factors for the development of complications. Surgical access for IAT includes intra-operative, immediate or delayed infusion via an "exteriorized" vein, and radiological percutaneous approaches. Delayed infusion can be combined with pancreas preservation techniques prior to islet isolation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Impact of adverse pancreatic injury at surgical procurement upon islet isolation outcome.

PubMed

Andres, Axel; Kin, Tatsuya; O'Gorman, Doug; Bigam, David; Kneteman, Norman; Senior, Peter; Shapiro, Am James

2014-11-01

The consequence of a pancreas injury during the procurement for islet isolation purpose is unknown. The goal of this work was to assess the injuries of the pancreata procured for islet isolation, and to determine their effect on the islet yield. Between January 2007 and October 2013, we prospectively documented every injury of the pancreata processed in our centre for islet isolation. Injuries involving the main duct were classified as major, the others as minor. Donors' characteristics and islet yields were compared between the groups of injuries. A pancreas injury was identified in 42 of 452 pancreata received for islet isolation (9.3%). In 15 cases, the injury was major (3.3% of all pancreata). Although a minor injury did not affect the islet yield, a major injury was significantly associated with unfavourable outcomes (postpurification mean islet equivalent of 364 ± 181, 405 ± 190 and 230 ± 115 × 10(3) for absence of injury, minor injury and major injury, respectively). A major injury was significantly more prevalent in lean and short donors. We recommend assessing the quality of the pancreas in the islet isolation centre before starting the isolation procedure. Each centre should determine its own policy based on its financial resources and on the wait list. © 2014 Steunstichting ESOT.

Consequences of Exposure to Light at Night on the Pancreatic Islet Circadian Clock and Function in Rats

PubMed Central

Qian, Jingyi; Block, Gene D.; Colwell, Christopher S.; Matveyenko, Aleksey V.

2013-01-01

There is a correlation between circadian disruption, type 2 diabetes mellitus (T2DM), and islet failure. However, the mechanisms underlying this association are largely unknown. Pancreatic islets express self-sustained circadian clocks essential for proper β-cell function and survival. We hypothesized that exposure to environmental conditions associated with disruption of circadian rhythms and susceptibility to T2DM in humans disrupts islet clock and β-cell function. To address this hypothesis, we validated the use of Per-1:LUC transgenic rats for continuous longitudinal assessment of islet circadian clock function ex vivo. Using this methodology, we subsequently examined effects of the continuous exposure to light at night (LL) on islet circadian clock and insulin secretion in vitro in rat islets. Our data show that changes in the light–dark cycle in vivo entrain the phase of islet clock transcriptional oscillations, whereas prolonged exposure (10 weeks) to LL disrupts islet circadian clock function through impairment in the amplitude, phase, and interislet synchrony of clock transcriptional oscillations. We also report that exposure to LL leads to diminished glucose-stimulated insulin secretion due to a decrease in insulin secretory pulse mass. Our studies identify potential mechanisms by which disturbances in circadian rhythms common to modern life can predispose to islet failure in T2DM. PMID:23775768

Important role of heparan sulfate in postnatal islet growth and insulin secretion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Iwao; Noguchi, Naoya; Nata, Koji

2009-05-22

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet {beta}-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in {beta}-cells. These mice exhibited abnormal islet morphology with reduced {beta}-cell proliferation after 1 week of age and glucosemore » intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.« less

Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene.

PubMed

Hrytsenko, Olga; Pohajdak, Bill; Wright, James R

2016-07-03

Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish.

Ancestral genomic duplication of the insulin gene in tilapia: An analysis of possible implications for clinical islet xenotransplantation using donor islets from transgenic tilapia expressing a humanized insulin gene

PubMed Central

Hrytsenko, Olga; Pohajdak, Bill; Wright, James R.

2016-01-01

ABSTRACT Tilapia, a teleost fish, have multiple large anatomically discrete islets which are easy to harvest, and when transplanted into diabetic murine recipients, provide normoglycemia and mammalian-like glucose tolerance profiles. Tilapia insulin differs structurally from human insulin which could preclude their use as islet donors for xenotransplantation. Therefore, we produced transgenic tilapia with islets expressing a humanized insulin gene. It is now known that fish genomes may possess an ancestral duplication and so tilapia may have a second insulin gene. Therefore, we cloned, sequenced, and characterized the tilapia insulin 2 transcript and found that its expression is negligible in islets, is not islet-specific, and would not likely need to be silenced in our transgenic fish. PMID:27222321

Impact of Procedure-Related Complications on Long-term Islet Transplantation Outcome.

PubMed

Caiazzo, Robert; Vantyghem, Marie-Christine; Raverdi, Violeta; Bonner, Caroline; Gmyr, Valery; Defrance, Frederique; Leroy, Clara; Sergent, Geraldine; Hubert, Thomas; Ernst, Oliver; Noel, Christian; Kerr-Conte, Julie; Pattou, François

2015-05-01

Pancreatic islet transplantation offers a promising biotherapy for the treatment of type 1 diabetes, but this procedure has met significant challenges over the years. One such challenge is to address why primary graft function still remains inconsistent after islet transplantation. Several variables have been shown to affect graft function, but the impact of procedure-related complications on primary and long-term graft functions has not yet been explored. Twenty-six patients with established type 1 diabetes were included in this study. Each patient had two to three intraportal islet infusions to obtain 10,000 islet equivalent (IEQ)/kg in body weight, equaling a total of 68 islet infusions. Islet transplantation consisted of three sequential fresh islet infusions within 3 months. Islet infusions were performed surgically or under ultrasound guidance, depending on patient morphology, availability of the radiology suite, and patient medical history. Prospective assessment of adverse events was recorded and graded using "Common Terminology Criteria for adverse events in Trials of Adult Pancreatic Islet Transplantation." There were no deaths or patients dropouts. Early complications occurred in nine of 68 procedures. β score 1 month after the last graft and optimal graft function (β score ≥7) rate were significantly lower in cases of procedure-related complications (P = 0.02, P = 0.03). Procedure-related complications negatively impacted graft function (P = 0.009) and was an independent predictive factor of long-term graft survival (P = 0.033) in multivariate analysis. Complications occurring during radiologic or surgical intraportal islet transplantation significantly impair primary graft function and graft survival regardless of their severity.

Assessment of DNA synthesis in Islet-1{sup +} cells in the adult murine heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weinberger, Florian, E-mail: f.weinberger@uke.de; Mehrkens, Dennis, E-mail: dennis.mehrkens@uk-koeln.de; Starbatty, Jutta, E-mail: starbatty@uke.uni-hamburg.de

Highlights: • Islet-1 was expressed in the adult heart. • Islet-1-positive cells did not proliferate in the adult heart. • Sinoatrial node cells did not proliferate in the adult heart. - Abstract: Rationale: Islet-1 positive (Islet-1{sup +}) cardiac progenitor cells give rise to the right ventricle, atria and outflow tract during murine cardiac development. In the adult heart Islet-1 expression is limited to parasympathetic neurons, few cardiomyocytes, smooth muscle cells, within the proximal aorta and pulmonary artery and sinoatrial node cells. Its role in these cells is unknown. Here we tested the hypothesis that Islet-1{sup +} cells retain proliferative activitymore » and may therefore play a role in regenerating specialized regions in the heart. Methods and results: DNA synthesis was analyzed by the incorporation of tritiated thymidine ({sup 3}H-thymidine) in Isl-1-nLacZ mice, a transgenic model with an insertion of a nuclear beta-galactosidase in the Islet-1 locus. Mice received daily injections of {sup 3}H-thymidine for 5 days. DNA synthesis was visualized throughout the heart by dipping autoradiography of cryosections. Colocalization of an nLacZ-signal and silver grains would indicate DNA synthesis in Islet-1{sup +} cells. Whereas Islet{sup −} non-myocyte nuclei were regularly marked by accumulation of silver grains, colocalization with nLacZ-signals was not detected in >25,000 cells analyzed. Conclusions: Islet-1{sup +} cells are quiescent in the adult heart, suggesting that, under normal conditions, even pacemaking cells do not proliferate at higher rates than normal cardiac myocytes.« less

Incorporation of Bone Marrow Cells in Pancreatic Pseudoislets Improves Posttransplant Vascularization and Endocrine Function

PubMed Central

Wittig, Christine; Laschke, Matthias W.; Scheuer, Claudia; Menger, Michael D.

2013-01-01

Failure of revascularization is known to be the major reason for the poor outcome of pancreatic islet transplantation. In this study, we analyzed whether pseudoislets composed of islet cells and bone marrow cells can improve vascularization and function of islet transplants. Pancreatic islets isolated from Syrian golden hamsters were dispersed into single cells for the generation of pseudoislets containing 4×103 cells. To create bone marrow cell-enriched pseudoislets 2×103 islet cells were co-cultured with 2×103 bone marrow cells. Pseudoislets and bone marrow cell-enriched pseudoislets were transplanted syngeneically into skinfold chambers to study graft vascularization by intravital fluorescence microscopy. Native islet transplants served as controls. Bone marrow cell-enriched pseudoislets showed a significantly improved vascularization compared to native islets and pseudoislets. Moreover, bone marrow cell-enriched pseudoislets but not pseudoislets normalized blood glucose levels after transplantation of 1000 islet equivalents under the kidney capsule of streptozotocin-induced diabetic animals, although the bone marrow cell-enriched pseudoislets contained only 50% of islet cells compared to pseudoislets and native islets. Fluorescence microscopy of bone marrow cell-enriched pseudoislets composed of bone marrow cells from GFP-expressing mice showed a distinct fraction of cells expressing both GFP and insulin, indicating a differentiation of bone marrow-derived cells to an insulin-producing cell-type. Thus, enrichment of pseudoislets by bone marrow cells enhances vascularization after transplantation and increases the amount of insulin-producing tissue. Accordingly, bone marrow cell-enriched pseudoislets may represent a novel approach to increase the success rate of islet transplantation. PMID:23875013

HMGB1 modulation in pancreatic islets using a cell-permeable A-box fragment.

PubMed

Hwang, Yong Hwa; Kim, Min Jun; Lee, Yong-Kyu; Lee, Minhyung; Lee, Dong Yun

2017-01-28

Although pancreatic islet implantation is an attractive strategy for curing diabetes mellitus, implanted cells are immunologically eliminated due to early islet graft loss. One of main issues in early islet graft loss is the secretion of high-mobility group-box-1 (HMGB1) protein from the damaged islet cells, which is known as a cytokine-like factor. Therefore, regulating the activity of HMGB1 protein offers an alternative strategy for improving outcomes of islet cell therapy. To this end, we first demonstrated that HMGB1 protein could be bound to its A-box fragment (HMGB1 A-box) with higher binding affinity, resembling anti-HMGB1 antibody. To be used as a pharmaceutical protein ex vivo, TAT-labeled HMGB1 A-box-His 6 (TAT-HMGB1A) was structurally modified for cellular membrane penetration. TAT-HMGB1A significantly reduced secretion of endogenous HMGB1 protein through interaction in the cytosol without any damage to the viability or functionality of the islets. When TAT-HMGB1A-treated islets were implanted into diabetic nude mice, they completely cured diabetes, as evidenced by stable blood glucose level. TAT-HMGB1A treatment could also reduce the marginal islet mass needed to cure diabetes. Furthermore, TAT-HMGB1A positively protected xenotransplanted islets from xenogeneic immune reactions. Collectively, cell-penetrable TAT-HMGB1A could be used to modulate HMGB1 activity to increase successful outcomes of ex vivo pancreatic islet cell therapy. Copyright © 2016 Elsevier B.V. All rights reserved.

A Low-Oxygenated Subpopulation of Pancreatic Islets Constitutes a Functional Reserve of Endocrine Cells

PubMed Central

Olsson, Richard; Carlsson, Per-Ola

2011-01-01

OBJECTIVE The blood perfusion of pancreatic islets is highly variable and tightly regulated by the blood glucose concentration. Thus, oxygen levels are considered crucial for islet metabolism and function. Although islet oxygenation has been extensively studied in vitro, little is known about it in vivo. The current study aimed to investigate the oxygenation of the endocrine pancreas in vivo. RESEARCH DESIGN AND METHODS The reductive metabolism of 2-nitroimidazoles, such as pimonidazole, has previously been extensively used in studies of oxygen metabolism both in vitro and in vivo. At tissue oxygen levels <10 mmHg, pimonidazole accumulates intracellularly and may thereafter be detected by means of immunohistochemistry. Islet oxygenation was investigated in normal, 60% partially pancreatectomized, as well as whole-pancreas–transplanted rats. Moreover, leucine-dependent protein biosynthesis was performed using autoradiography to correlate islet oxygenation with metabolic activity. RESULTS In vivo, 20–25% of all islets in normal rats showed low oxygenation (pO2 <10 mmHg). Changes in the islet mass, by means of whole-pancreas transplantation, doubled the fraction of low-oxygenated islets in the endogenous pancreas of transplanted animals, whereas this fraction almost completely disappeared after a 60% partial pancreatectomy. Moreover, oxygenation was related to metabolism, since well-oxygenated islets in vivo had 50% higher leucine-dependent protein biosynthesis, which includes (pro)insulin biosynthesis. CONCLUSIONS The current study suggests a novel subpopulation of dormant low-oxygenated islets, which seems to constitute a functional reserve of endocrine cells. This study establishes a novel perspective on the use of the endocrine pancreas in glucose homeostasis. PMID:21788581

Redox-Dependent Inflammation in Islet Transplantation Rejection

PubMed Central

Barra, Jessie M.; Tse, Hubert M.

2018-01-01

Type 1 diabetes is an autoimmune disease that results in the progressive destruction of insulin-producing pancreatic β-cells inside the islets of Langerhans. The loss of this vital population leaves patients with a lifelong dependency on exogenous insulin and puts them at risk for life-threatening complications. One method being investigated to help restore insulin independence in these patients is islet cell transplantation. However, challenges associated with transplant rejection and islet viability have prevented long-term β-cell function. Redox signaling and the production of reactive oxygen species (ROS) by recipient immune cells and transplanted islets themselves are key players in graft rejection. Therefore, dissipation of ROS generation is a viable intervention that can protect transplanted islets from immune-mediated destruction. Here, we will discuss the newly appreciated role of redox signaling and ROS synthesis during graft rejection as well as new strategies being tested for their efficacy in redox modulation during islet cell transplantation. PMID:29740396

Current Status of Immunomodulatory and Cellular Therapies in Preclinical and Clinical Islet Transplantation

PubMed Central

Chhabra, Preeti; Brayman, Kenneth L.

2011-01-01

Clinical islet transplantation is a β-cell replacement strategy that represents a possible definitive intervention for patients with type 1 diabetes, offering substantial benefits in terms of lowering daily insulin requirements and reducing incidences of debilitating hypoglycemic episodes and unawareness. Despite impressive advances in this field, a limiting supply of islets, inadequate means for preventing islet rejection, and the deleterious diabetogenic and nephrotoxic side effects associated with chronic immunosuppressive therapy preclude its wide-spread applicability. Islet transplantation however allows a window of opportunity for attempting various therapeutic manipulations of islets prior to transplantation aimed at achieving superior transplant outcomes. In this paper, we will focus on the current status of various immunosuppressive and cellular therapies that promote graft function and survival in preclinical and clinical islet transplantation with special emphasis on the tolerance-inducing capacity of regulatory T cells as well as the β-cells regenerative capacity of stem cells. PMID:22046502

Desnutrin/ATGL activates PPARδ to promote mitochondrial function for insulin secretion in islet β cells.

PubMed

Tang, Tianyi; Abbott, Marcia J; Ahmadian, Maryam; Lopes, Andressa B; Wang, Yuhui; Sul, Hei Sook

2013-12-03

Excessive caloric intake leading to obesity is associated with insulin resistance and dysfunction of islet β cells. High-fat feeding decreases desnutrin (also called ATGL/PNPLA2) levels in islets. Here we show that desnutrin ablation via RIP-Cre (βKO) or RIP-CreER results in hyperglycemia with impaired glucose-stimulated insulin secretion (GSIS). Due to decreased lipolysis, islets have higher TAG content but lower free FA levels. βKO islets exhibit impaired mitochondrial respiration and lower production of ATP required for GSIS, along with decreased expression of PPARδ target genes involved in mitochondrial oxidation. Furthermore, synthetic PPARδ, but not PPARα, agonist restores GSIS and expression of mitochondrial oxidative genes in βKO mice, revealing that desnutrin-catalyzed lipolysis generates PPARδ ligands. Finally, adenoviral expression of desnutrin in βKO islets restores all defects of βKO islet phenotype and function, including GSIS and mitochondrial defects, demonstrating the critical role of the desnutrin-PPARδ-mitochondrial oxidation axis in regulating islet β cell GSIS. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of Trasina, an Ayurvedic herbal formulation, on pancreatic islet superoxide dismutase activity in hyperglycaemic rats.

PubMed

Bhattacharya, S K; Satyan, K S; Chakrabarti, A

1997-03-01

Diabetes mellitus was induced in male CF strain rats by streptozotocin (STZ) and hyperglycaemia and superoxide dismutase (SOD) activity of pancreatic islet cells was assessed on days 7, 14, 21 and 28. STZ induced significant hyperglycaemia and a concomitant decrease in islet cell SOD activity. Transina (TR), an Ayurvedic herbal formulation comprising of Withania somnifera, Tinospora cordifolia, Eclipta alba, Ocimum sanctum, Picrorrhiza kurroa and shilajit, had little per se effect on blood sugar concentrations and islet SOD activity in euglycaemic rats, in the doses of 100 and 200 mg/kg, p.o. administered once daily for 28 days. However, these doses of TR induced a dose- related decrease in STZ hyperglycaemia and attenuation of STZ induced decrease in islet SOD activity. The results indicate that the earlier reported anti-hyperglycaemic effect of TR may be due to pancreatic islet free radical scavenging activity, the hyperglycaemic activity of STZ being the consequence of decrease in islet SOD activity leading to the accumulation of degenerative oxidative free radicals in islet beta-cells.

Tissue-specific exosome biomarkers for noninvasively monitoring immunologic rejection of transplanted tissue

PubMed Central

Korutla, Laxminarayana; Habertheuer, Andreas; Yu, Ming; Rostami, Susan; Yuan, Chao-Xing; Reddy, Sanjana; Korutla, Varun; Koeberlein, Brigitte; Trofe-Clark, Jennifer; Rickels, Michael R.; Naji, Ali

2017-01-01

In transplantation, there is a critical need for noninvasive biomarker platforms for monitoring immunologic rejection. We hypothesized that transplanted tissues release donor-specific exosomes into recipient circulation and that the quantitation and profiling of donor intra-exosomal cargoes may constitute a biomarker platform for monitoring rejection. Here, we have tested this hypothesis in a human-into-mouse xenogeneic islet transplant model and validated the concept in clinical settings of islet and renal transplantation. In the xenogeneic model, we quantified islet transplant exosomes in recipient blood over long-term follow-up using anti-HLA antibody, which was detectable only in xenoislet recipients of human islets. Transplant islet exosomes were purified using anti-HLA antibody–conjugated beads, and their cargoes contained the islet endocrine hormone markers insulin, glucagon, and somatostatin. Rejection led to a marked decrease in transplant islet exosome signal along with distinct changes in exosomal microRNA and proteomic profiles prior to appearance of hyperglycemia. In the clinical settings of islet and renal transplantation, donor exosomes with respective tissue specificity for islet β cells and renal epithelial cells were reliably characterized in recipient plasma over follow-up periods of up to 5 years. Collectively, these findings demonstrate the biomarker potential of transplant exosome characterization for providing a noninvasive window into the conditional state of transplant tissue. PMID:28319051

Teucrium polium complex with molybdate enhance cultured islets secretory function.

PubMed

Mohseni Salehi Monfared, Seyed Sajad; Pournourmohammadi, Shirin

2010-02-01

Islet transplantation has become a promising treatment in the therapy of type 1 diabetes. Its function improvement, after isolation and before transplantation, is crucial because of their loss both in number and function of islets after isolation procedures. Trace elements sodium orthovanadate (SOV) and sodium molybdate (SM), as well as medicinal plant Teucrium polium L. (TP), showed and possessed high beneficial antioxidative potential and even hypoglycemic properties via their effect on islets. We evaluated the effect of these components in combination on cultured islet function in order to improve pancreatic islet transplantation. Rat pancreatic islets were cultured for 24 h then incubated with different concentrations of TP (0.01 and 0.1 mg/mL) alone and in combination with SOV (1 mM) or SM (1 mM). Insulin concentration in buffer media was measured as islet secretory function. Administration of TP (0.01 mg/mL), SM, and SOV alone or in combination with each other significantly increased insulin secretion at high glucose concentration (16.7 mM); insulin secretion was significantly greater in the group containing both TP and SM than other treated groups (p < 0.05). The combination of the mentioned trace elements especially molybdate with TP could improve islet cells function before transplantation.

Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.

PubMed

Wen, Di; Peng, Yang; Liu, Di; Weizmann, Yossi; Mahato, Ram I

2016-09-28

Human bone marrow mesenchymal stem cells (hBMSCs) and their exosomes can suppress immune reaction and deliver small RNAs. Thus, they may improve islet transplantation by delivering small RNAs for promoting islet function and inhibiting immune rejection. Here, we proposed an hBMSC and its exosome-based therapy to overcome immune rejection and poor islet function, both of which hinder the success of islet transplantation. We found overexpressed siFas and anti-miR-375 in plasmid encoding shFas and anti-miR-375 transfected hBMSC-derived exosomes, which silenced Fas and miR-375 of human islets and improved their viability and function against inflammatory cytokines. This plasmid transfected hBMSCs downregulated Fas and miR-375 of human islets in a humanized NOD scid gamma (NSG) mouse model, whose immune reaction was inhibited by injecting hBMSC and peripheral blood mononuclear cell (PBMC) co-cultured exosomes. These exosomes suppressed immune reaction by inhibiting PBMC proliferation and enhancing regulatory T cell (Treg) function. Collectively, our studies elucidated the mechanisms of RNA delivery from hBMSCs to human islets and the immunosuppressive effect of hBMSC and peripheral blood mononuclear cell co-cultured exosomes for improving islet transplantation. Copyright © 2016 Elsevier B.V. All rights reserved.

Successful β cells islet regeneration in streptozotocin-induced diabetic baboons using ultrasound-targeted microbubble gene therapy with cyclinD2/CDK4/GLP1

PubMed Central

Chen, Shuyuan; Bastarrachea, Raul A; Roberts, Brad J; Voruganti, V Saroja; Frost, Patrice A; Nava-Gonzalez, Edna J; Arriaga-Cazares, Hector E; Chen, Jiaxi; Huang, Pintong; DeFronzo, Ralph A; Comuzzie, Anthony G; Grayburn, Paul A

2014-01-01

Both major forms of diabetes mellitus (DM) involve β-cell destruction and dysfunction. New treatment strategies have focused on replenishing the deficiency of β-cell mass common to both major forms of diabetes by islet transplantation or β-cell regeneration. The pancreas, not the liver, is the ideal organ for islet regeneration, because it is the natural milieu for islets. Since islet mass is known to increase during obesity and pregnancy, the concept of stimulating pancreatic islet regeneration in vivo is both rational and physiologic. This paper proposes a novel approach in which non-viral gene therapy is targeted to pancreatic islets using ultrasound targeted microbubble destruction (UTMD) in a non-human primate model (NHP), the baboon. Treated baboons received a gene cocktail comprised of cyclinD2, CDK, and GLP1, which in rats results in robust and durable islet regeneration with normalization of blood glucose, insulin, and C-peptide levels. We were able to generate important preliminary data indicating that gene therapy by UTMD can achieve in vivo normalization of the intravenous (IV) glucose tolerance test (IVGTT) curves in STZ hyperglycemic-induced conscious tethered baboons. Immunohistochemistry clearly demonstrated evidence of islet regeneration and restoration of β-cell mass. PMID:24553120

Successful β cells islet regeneration in streptozotocin-induced diabetic baboons using ultrasound-targeted microbubble gene therapy with cyclinD2/CDK4/GLP1.

PubMed

Chen, Shuyuan; Bastarrachea, Raul A; Roberts, Brad J; Voruganti, V Saroja; Frost, Patrice A; Nava-Gonzalez, Edna J; Arriaga-Cazares, Hector E; Chen, Jiaxi; Huang, Pintong; DeFronzo, Ralph A; Comuzzie, Anthony G; Grayburn, Paul A

2014-01-01

Both major forms of diabetes mellitus (DM) involve β-cell destruction and dysfunction. New treatment strategies have focused on replenishing the deficiency of β-cell mass common to both major forms of diabetes by islet transplantation or β-cell regeneration. The pancreas, not the liver, is the ideal organ for islet regeneration, because it is the natural milieu for islets. Since islet mass is known to increase during obesity and pregnancy, the concept of stimulating pancreatic islet regeneration in vivo is both rational and physiologic. This paper proposes a novel approach in which non-viral gene therapy is targeted to pancreatic islets using ultrasound targeted microbubble destruction (UTMD) in a non-human primate model (NHP), the baboon. Treated baboons received a gene cocktail comprised of cyclinD2, CDK, and GLP1, which in rats results in robust and durable islet regeneration with normalization of blood glucose, insulin, and C-peptide levels. We were able to generate important preliminary data indicating that gene therapy by UTMD can achieve in vivo normalization of the intravenous (IV) glucose tolerance test (IVGTT) curves in STZ hyperglycemic-induced conscious tethered baboons. Immunohistochemistry clearly demonstrated evidence of islet regeneration and restoration of β-cell mass.

Endothelial chimerism and vascular sequestration protect pancreatic islet grafts from antibody-mediated rejection

PubMed Central

Chen, Chien-Chia; Pouliquen, Eric; Broisat, Alexis; Andreata, Francesco; Racapé, Maud; Bruneval, Patrick; Kessler, Laurence; Ahmadi, Mitra; Bacot, Sandrine; Saison-Delaplace, Carole; Marcaud, Marina; Van Huyen, Jean-Paul Duong; Loupy, Alexandre; Villard, Jean; Demuylder-Mischler, Sandrine; Morelon, Emmanuel; Tsai, Meng-Kun; Kolopp-Sarda, Marie-Nathalie; Koenig, Alice; Mathias, Virginie; Ghezzi, Catherine; Dubois, Valerie; Defrance, Thierry

2017-01-01

Humoral rejection is the most common cause of solid organ transplant failure. Here, we evaluated a cohort of 49 patients who were successfully grafted with allogenic islets and determined that the appearance of donor-specific anti-HLA antibodies (DSAs) did not accelerate the rate of islet graft attrition, suggesting resistance to humoral rejection. Murine DSAs bound to allogeneic targets expressed by islet cells and induced their destruction in vitro; however, passive transfer of the same DSAs did not affect islet graft survival in murine models. Live imaging revealed that DSAs were sequestrated in the circulation of the recipients and failed to reach the endocrine cells of grafted islets. We used murine heart transplantation models to confirm that endothelial cells were the only accessible targets for DSAs, which induced the development of typical microvascular lesions in allogeneic transplants. In contrast, the vasculature of DSA-exposed allogeneic islet grafts was devoid of lesions because sprouting of recipient capillaries reestablished blood flow in grafted islets. Thus, we conclude that endothelial chimerism combined with vascular sequestration of DSAs protects islet grafts from humoral rejection. The reduced immunoglobulin concentrations in the interstitial tissue, confirmed in patients, may have important implications for biotherapies such as vaccines and monoclonal antibodies. PMID:29202467

Autologous Mesenchymal Stem Cell and Islet Cotransplantation: Safety and Efficacy.

PubMed

Wang, Hongjun; Strange, Charlie; Nietert, Paul J; Wang, Jingjing; Turnbull, Taylor L; Cloud, Colleen; Owczarski, Stefanie; Shuford, Betsy; Duke, Tara; Gilkeson, Gary; Luttrell, Louis; Hermayer, Kathie; Fernandes, Jyotika; Adams, David B; Morgan, Katherine A

2018-01-01

Islet engraftment after transplantation is impaired by high rates of islet/β cell death caused by cellular stressors and poor graft vascularization. We studied whether cotransplantation of ex vivo expanded autologous bone marrow-derived mesenchymal stem cells (MSCs) with islets is safe and beneficial in chronic pancreatitis patients undergoing total pancreatectomy with islet autotransplantation. MSCs were harvested from the bone marrow of three islet autotransplantation patients and expanded at our current Good Manufacturing Practices (cGMP) facility. On the day of islet transplantation, an average dose of 20.0 ± 2.6 ×10 6 MSCs was infused with islets via the portal vein. Adverse events and glycemic control at baseline, 6, and 12 months after transplantation were compared with data from 101 historical control patients. No adverse events directly related to the MSC infusions were observed. MSC patients required lower amounts of insulin during the peritransplantation period (p = .02 vs. controls) and had lower 12-month fasting blood glucose levels (p = .02 vs. controls), smaller C-peptide declines over 6 months (p = .01 vs. controls), and better quality of life compared with controls. In conclusion, our pilot study demonstrates that autologous MSC and islet cotransplantation may be a safe and potential strategy to improve islet engraftment after transplantation. (Clinicaltrials.gov registration number: NCT02384018). Stem Cells Translational Medicine 2018;7:11-19. © 2017 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Islet grafting and imaging in a bioengineered intramuscular space.

PubMed

Witkowski, Piotr; Sondermeijer, Hugo; Hardy, Mark A; Woodland, David C; Lee, Keagan; Bhagat, Govind; Witkowski, Kajetan; See, Fiona; Rana, Abbas; Maffei, Antonella; Itescu, Silviu; Harris, Paul E

2009-11-15

Because the hepatic portal system may not be the optimal site for islet transplantation, several extrahepatic sites have been studied. Here, we examine an intramuscular transplantation site, bioengineered to better support islet neovascularization, engraftment, and survival, and we demonstrate that at this novel site, grafted beta cell mass may be quantitated in a real-time noninvasive manner by positron emission tomography (PET) imaging. Streptozotocin-induced rats were pretreated intramuscularly with a biocompatible angiogenic scaffold received syngeneic islet transplants 2 weeks later. The recipients were monitored serially by blood glucose and glucose tolerance measurements and by PET imaging of the transplant site with [11C] dihydrotetrabenazine. Parallel histopathologic evaluation of the grafts was performed using insulin staining and evaluation of microvasularity. Reversal of hyperglycemia by islet transplantation was most successful in recipients pretreated with bioscaffolds containing angiogenic factors when compared with those who received no bioscaffolds or bioscaffolds not treated with angiogenic factors. PET imaging with [11C] dihydrotetrabenazine, insulin staining, and microvascular density patterns were consistent with islet survival, increased levels of angiogenesis, and with reversal of hyperglycemia. Induction of increased neovascularization at an intramuscular site significantly improves islet transplant engraftment and survival compared with controls. The use of a nonhepatic transplant site may avoid intrahepatic complications and permit the use of PET imaging to measure and follow transplanted beta cell mass in real time. These findings have important implications for effective islet implantation outside of the liver and offer promising possibilities for improving islet survival, monitoring, and even prevention of islet loss.

An effective purification method using large bottles for human pancreatic islet isolation

PubMed Central

Shimoda, Masayuki; Itoh, Takeshi; Iwahashi, Shuichi; Takita, Morihito; Sugimoto, Koji; Kanak, Mazhar A.; Chujo, Daisuke; Naziruddin, Bashoo; Levy, Marlon F.; Grayburn, Paul A.; Matsumoto, Shinichi

2012-01-01

The purification process is one of the most difficult procedures in pancreatic islet isolation. It was demonstrated that the standard purification method using a COBE 2991 cell processor with Ficoll density gradient solution harmed islets mechanically by high shear force. We reported that purification using large bottles with a lower viscosity gradient solution could improve the efficacy of porcine islet purification. In this study, we examined whether the new bottle purification method could improve the purification of human islets. Nine human pancreata from brain-dead donors were used. After pancreas digestion, the digested tissue was divided into three groups. Each group was purified by continuous density gradient using ET-Kyoto and iodixanol gradient solution with either the standard COBE method (COBE group) or the top loading (top group) or bottom loading (bottom group) bottle purification methods. Islet yield, purity, recovery rate after purification, and in vitro and in vivo viability were compared. Islet yield per pancreas weight (IE/g) and the recovery rate in the top group were significantly higher than in the COBE and bottom groups. Furthermore, the average size of purified islets in the top group was significantly larger than in the COBE group, which indicated that the bottle method could reduce the shear force to the islets. In vivo viability was also significantly higher in the top group compared with the COBE group. In conclusion, the top-loading bottle method could improve the quality and quantity of human islets after purification. PMID:23221740

The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans

PubMed Central

Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

2015-01-01

Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets. PMID:26266953

The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans.

PubMed

Striegel, Deborah A; Hara, Manami; Periwal, Vipul

2015-08-01

Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets.

Characterization of the Mouse Pancreatic Islet Proteome and Comparative Analysis with Other Mouse Tissues

PubMed Central

Petyuk, Vladislav A.; Qian, Wei-Jun; Hinault, Charlotte; Gritsenko, Marina A.; Singhal, Mudita; Monroe, Matthew E.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.

2009-01-01

The pancreatic islets of Langerhans, and especially the insulin-producing beta cells, play a central role in the maintenance of glucose homeostasis. Alterations in the expression of multiple proteins in the islets that contribute to the maintenance of islet function are likely to underlie the pathogenesis of type 2 diabetes. To identify proteins that constitute the islet proteome, we provide the first comprehensive proteomic characterization of pancreatic islets for mouse, the most commonly used animal model in diabetes research. Using strong cation exchange fractionation coupled with reversed phase LC-MS/MS we report the confident identification of 17,350 different tryptic peptides covering 2,612 proteins having at least two unique peptides per protein. The dataset also identified ~60 post-translationally modified peptides including oxidative modifications and phosphorylation. While many of the identified phosphorylation sites corroborate those previously known, the oxidative modifications observed on cysteinyl residues reveal potentially novel information suggesting a role for oxidative stress in islet function. Comparative analysis with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 133 proteins predominantly expressed in pancreatic islets. This unique set of proteins, in addition to those with known functions such as peptide hormones secreted from the islets, contains several proteins with as yet unknown functions. The mouse islet protein and peptide database accessible at http://ncrr.pnl.gov, provides an important reference resource for the research community to facilitate research in the diabetes and metabolism fields. PMID:18570455

Glucose-dependent blood flow dynamics in murine pancreatic islets in vivo

PubMed Central

Nyman, Lara R.; Ford, Eric

2010-01-01

Pancreatic islets are highly vascularized and arranged so that regions containing β-cells are distinct from those containing other cell types. Although islet blood flow has been studied extensively, little is known about the dynamics of islet blood flow during hypoglycemia or hyperglycemia. To investigate changes in islet blood flow as a function of blood glucose level, we clamped blood glucose sequentially at hyperglycemic (∼300 mg/dl or 16.8 mM) and hypoglycemic (∼50 mg/dl or 2.8 mM) levels while simultaneously imaging intraislet blood flow in mouse models that express green fluorescent protein in the β-cells or yellow fluorescent protein in the α-cells. Using line scanning confocal microscopy, in vivo blood flow was assayed after intravenous injection of fluorescent dextran or sulforhodamine-labeled red blood cells. Regardless of the sequence of hypoglycemia and hyperglycemia, islet blood flow is faster during hyperglycemia, and apparent blood volume is greater during hyperglycemia than during hypoglycemia. However, there is no change in the order of perfusion of different islet endocrine cell types in hypoglycemia compared with hyperglycemia, with the islet core of β-cells usually perfused first. In contrast to the results in islets, there was no significant difference in flow rate in the exocrine pancreas during hyperglycemia compared with hypoglycemia. These results indicate that glucose differentially regulates blood flow in the pancreatic islet vasculature independently of blood flow in the rest of the pancreas. PMID:20071562

Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

PubMed Central

Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

2017-01-01

Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

α-1 Antitrypsin Enhances Islet Engraftment by Suppression of Instant Blood-Mediated Inflammatory Reaction.

PubMed

Wang, Jingjing; Sun, Zhen; Gou, Wenyu; Adams, David B; Cui, Wanxing; Morgan, Katherine A; Strange, Charlie; Wang, Hongjun

2017-04-01

Islet cell transplantation has limited effectiveness because of an instant blood-mediated inflammatory reaction (IBMIR) that occurs immediately after cell infusion and leads to dramatic β-cell death. In intraportal islet transplantation models using mouse and human islets, we demonstrated that α-1 antitrypsin (AAT; Prolastin-C), a serine protease inhibitor used for the treatment of AAT deficiency, inhibits IBMIR and cytokine-induced inflammation in islets. In mice, more diabetic recipients reached normoglycemia after intraportal islet transplantation when they were treated with AAT compared with mice treated with saline. AAT suppressed blood-mediated coagulation pathways by diminishing tissue factor production, reducing plasma thrombin-antithrombin complex levels and fibrinogen deposition on islet grafts, which correlated with less graft damage and apoptosis. AAT-treated mice showed reduced serum tumor necrosis factor-α levels, decreased lymphocytic infiltration, and decreased nuclear factor (NF)-κB activation compared with controls. The potent anti-inflammatory effect of AAT is possibly mediated by suppression of c-Jun N-terminal kinase (JNK) phosphorylation. Blocking JNK activation failed to further reduce cytokine-induced apoptosis in β-cells. Taken together, AAT significantly improves islet graft survival after intraportal islet transplantation by mitigation of coagulation in IBMIR and suppression of cytokine-induced JNK and NF-κB activation. AAT-based therapy has the potential to improve graft survival in human islet transplantation and other cellular therapies on the horizon. © 2017 by the American Diabetes Association.

Prior Surgery Determines Islet Yield and Insulin Requirement in Patients with Chronic Pancreatitis

PubMed Central

Wang, Hongjun; Desai, Krupa D; Dong, Huansheng; Owzarski, Stefanie; Romagnuolo, Joseph; Morgan, Katherine A; Adams, David B

2013-01-01

Background Total pancreatectomy with islet autotransplantation (TP-IAT) is safe and effective in the management of intractable pain associated with chronic pancreatitis (CP). Prevention of pancreatogenic diabetes after TP-IAT is related to islet yield from the diseased pancreas. The purpose of this study is to compare islet yield and insulin requirement in the 76 patients who underwent different surgical procedures prior to TP-IAT at the Medical University of South Carolina between the years 2009 to 2011. Methods Patients were grouped into four categories based on the operation they had before TP-IAT: transduodenal sphincteroplasty or no prior surgery (TDS/NPS, n=50), Whipple or Beger procedure (WB, n=14), distal pancreatectomy (DP, n=8) or lateral pancreaticojejunostomy (LPJ, n=4). Islets were harvested from pancreases of those patients at our cGMP facility. Total unpurified islets were transplanted into patients via portal vein infusion. Pancreatic fibrosis, islet yield, cell viability and insulin requirement were measured. Results The pancreases of TDS/NPS and WB patients were less fibrotic, and had higher islet yield compared to those who had DP or LPJ. Higher islet yield also correlated with a greater diabetes free rate and a lesser insulin requirement at the following intervals: pre-operative, post-operative and 6 months after TP-IAT. Conclusions Prior surgery is strongly correlated with the extent of pancreatic fibrosis, islet yield and insulin requirements in CP patients undergoing TP-IAT. The history of prior pancreatic resection and drainage procedures may be used to predict post-operative islet function and help to determine the optimal timing for TP-IAT in CP patients. PMID:23411743

Single-donor islet transplantation and long-term insulin independence in select patients with type 1 diabetes mellitus.

PubMed

Al-Adra, David P; Gill, Richdeep S; Imes, Sharleen; O'Gorman, Doug; Kin, Tatsuya; Axford, Sara J; Shi, Xinzhe; Senior, Peter A; Shapiro, A M James

2014-11-15

Islet transplantation is a recognized treatment option for select patients with type I diabetes mellitus. However, islet infusions from multiple donors are often required to achieve insulin independence. Ideally, insulin independence would be achieved routinely with only a single donor. Identification of factors associated with insulin independence after single-donor islet transplantation may help to select recipient-donor combinations with the highest probability of success. Subjects undergoing islet transplantation at a single center (Edmonton, Canada) between March 1999 and August 2013 were included. Recipient, donor, and transplant characteristics were collected and compared between recipients who became insulin independent after one islet transplantation and those who did not. Thirty-one patients achieved insulin independence after a single-donor islet transplantation, and 149 did not. Long-term insulin-free survival was not different between the groups. Factors significantly associated with single-donor success included recipient age, insulin requirement at baseline, donor weight, donor body mass index, islet transplant mass, and peritransplant heparin and insulin administration. On multivariate analysis, pretransplantation daily insulin requirements, the use of peritransplantation heparin and insulin infusions, and islet transplant mass remained significant. We have identified clinically relevant differences defining the achievement of insulin independence after single-donor transplantation. Based on these differences, a preoperative insulin requirement of less than 0.6 U/kg per day and receiving more than 5,646 islet equivalents (IEQ)/kg have a sensitivity of 84% and 71% and specificity of 50% and 50%, respectively, for insulin independence after single-donor islet transplantation. With ideal patient selection, this finding could potentially increase single-donor transplantation success and may be especially relevant for presensitized subjects or those who may subsequently require renal replacement.

Comparison of therapeutic characteristics of islet cell transplantation simultaneous with pancreatic mesenchymal stem cell transplantation in rats with Type 1 diabetes mellitus.

PubMed

Unsal, Ilknur Ozturk; Ginis, Zeynep; Pinarli, Ferda Alparslan; Albayrak, Aynur; Cakal, Erman; Sahin, Mustafa; Delibasi, Tuncay

2015-06-01

Although, pancreas islet call transplantation is a new, promising method for type 1 diabetic patients, it remains as an experimental procedure applied in selected patients. The present study aimed to investigate effect of pancreatic mesenchymal stem cell transplantation simultaneous with islet cell transplantation on islet liveliness and thus on the treatment of diabetes in type 1 diabetic rats. The study used Wistar Albino Rats and was performed in a total of four groups [control (G1), mesenchymal stem cell (G2), islet (G3) and islet + mesencymal stem cell (G4)] each including 8 rats. Blood glucose level of the rats, in which diabetes model has been created using streptozotocin, was measured after 72 h. Blood samples were obtained from the rats 30 days after transplantation and then, their livers and pancreases were kept in 10% formaldehyde and the experiment was ended. Following staining with H&E, they were morphologically evaluated under a light microscope. Change in mean blood glucose level was statistically significant in G3 and G4 versus G1 and G2 (p = 0.001, p < 0.001, p < 0.001, and p < 0.001 respectively). Histological examination revealed that mean number of islet cells in the pancreases of the rats was higher in G4; difference between the groups was statistically significant (p < 0.001). Transplantation of islet cells together with mesenchymal stem cells showed beneficial effects in terms of prolonging survival of islet grafts suggesting that transplantation of mesenchymal stem cells together with islet cells during clinical islet transplantation may be beneficial in increasing the number of noninsulin-dependent patients in Type 1 diabetes.

Effects of cholinergic m-receptor agonists on insulin release in islets from obese and lean mice of different ages: the importance of bicarbonate.

PubMed

Persson-Sjögren, Solveig; Lindström, Per

2004-11-01

Decreased beta-cell function is often observed in older individuals and may predispose to the development of type 2 diabetes. We have studied the age-related effects of M-receptor agonism on insulin release in islets isolated from female ob/ ob and lean mice. Islets were challenged with 11.1 or 16.7 mmol/L glucose in media with HCO3/CO2 (KRBH) or without (KRH). Acetylcholine (ACh) (10 micromol/L) increased glucose-induced insulin release in islets from 4- to 5-week-old ob/ob mice both in KRBH and KRH. In islets from 9- to 13-month-old ob/ob mice, 10 micromol/L ACh and 10 micromol/L carbachol enhanced insulin release in KRBH but not in KRH. ACh increased insulin release in islets from 4- to 5-week-old and 16-month-old lean mice incubated in KRH but not in islets from 24-month-old lean mice. The Na/H exchange inhibitor dimethylamiloride (100 micromol/L) did not affect insulin release stimulated by M-receptor agonists. Carbachol did not enhance glucose-induced insulin secretion in islets from 9- to 10-month-old ob/ob mice in the presence of low extracellular Na concentration. ACh stimulated cytoplasmic Ca mobilization in islets from 9- to 10-month-old mice also when bicarbonate was omitted. The results suggest that cholinergic signal transduction involving extracellular bicarbonate and Na is reduced with age in mouse pancreatic islets. Chronic hyperglycemia may add to the age-related decrease in M-receptor-mediated insulin release by affecting the buffering capacity of the islets through mechanisms other than amiloride-sensitive proton exchange.

Metabolic Profile of Pancreatic Acinar and Islet Tissue in Culture

PubMed Central

Suszynski, Thomas M.; Mueller, Kathryn; Gruessner, Angelika C.; Papas, Klearchos K.

2016-01-01

The amount and condition of exocrine impurities may affect the quality of islet preparations especially during culture. In this study, the objective was to determine the oxygen demandand viability of islet and acinar tissue post-isolation and whether they change disproportionately while in culture. We compare the OCR normalized to DNA (OCR/DNA, a measure of fractional viability in units nmol/min/mg DNA), and percent change in OCR and DNA recoveries between adult porcine islet and acinar tissue from the same preparation (paired) over a 6-9 days of standard culture. Paired comparisons were done to quantify differences in OCR/DNA between islet and acinar tissue from the same preparation, at specified time points during culture; the mean (± standard error) OCR/DNA was 74.0 (±11.7) units higher for acinar (vs. islet) tissue on the day of isolation (n=16, p<0.0001), but 25.7 (±9.4) units lower after 1 day (n=8, p=0.03), 56.6 (±11.5) units lower after 2 days (n=12, p=0.0004), and 65.9 (±28.7) units lower after 8 days (n=4, p=0.2) in culture. DNA and OCR recoveries decreased at different rates for acinar versus islet tissue over 6-9 days in culture (n=6). DNA recovery decreased to 24±7% for acinar and 75±8% for islets (p=0.002). Similarly, OCR recovery decreased to 16±3% for acinar and remained virtually constant for islets (p=0.005). Differences in the metabolic profile of acinarand islet tissue should be considered when culturing impure islet preparations. OCR-based measurements may help optimize pre-IT culture protocols. PMID:25131082

Role of T-cell-specific nuclear factor κB in islet allograft rejection.

PubMed

Porras, Delia Lozano; Wang, Ying; Zhou, Ping; Molinero, Luciana L; Alegre, Maria-Luisa

2012-05-27

Pancreatic islet transplantation has the potential to cure type 1 diabetes, a chronic lifelong disease, but its clinical applicability is limited by allograft rejection. Nuclear factor κB (NF-κB) is a transcription factor important for survival and differentiation of T cells. In this study, we tested whether NF-κB in T cells is required for the rejection of islet allografts. Mice expressing a superrepressor form of NF-κB selectively in T cells (IκBαΔN-Tg mice) with or without the antiapoptotic factor Bcl-xL, or mice with impaired T-cell receptor (TCR)- and B cell receptor-driven NF-κB activity (CARMA1-KO mice) were rendered diabetic and transplanted with islet allografts. Secondary skin transplantation in long-term acceptors of islet allografts was used to test for the development of donor-specific tolerance. Immune infiltration of the transplanted islets was examined by immunofluorescence. TCR-transgenic CD4 T cells were used to follow T-cell priming and differentiation. Islet allograft survival was prolonged in IκBαΔN-Tg mice, although the animals did not develop donor-specific tolerance. Reduced NF-κB activity did not prevent T-cell priming or differentiation but reduced survival of activated T cells, as transgenic expression of Bcl-xL restored islet allograft rejection in IκBαΔN-Tg mice. Abolishing TCR- and B cell receptor-driven activation of NF-κB selectively by CARMA1 deficiency prevented T-cell priming and islet allograft rejection. Our data suggest that T cell-NF-κB plays an important role in the rejection of islet allografts. Targeting NF-κB selectively in lymphocytes seems a promising approach to facilitate acceptance of transplanted islets.

Gap junctions and other mechanisms of cell-cell communication regulate basal insulin secretion in the pancreatic islet.

PubMed

Benninger, R K P; Head, W Steven; Zhang, Min; Satin, Leslie S; Piston, David W

2011-11-15

Cell-cell communication in the islet of Langerhans is important for the regulation of insulin secretion. Gap-junctions coordinate oscillations in intracellular free-calcium ([Ca(2+)](i)) and insulin secretion in the islet following elevated glucose. Gap-junctions can also ensure that oscillatory [Ca(2+)](i) ceases when glucose is at a basal levels. We determine the roles of gap-junctions and other cell-cell communication pathways in the suppression of insulin secretion under basal conditions. Metabolic, electrical and insulin secretion levels were measured from islets lacking gap-junction coupling following deletion of connexion36 (Cx36(-/-)), and these results were compared to those obtained using fully isolated β-cells. K(ATP) loss-of-function islets provide a further experimental model to specifically study gap-junction mediated suppression of electrical activity. In isolated β-cells or Cx36(-/-) islets, elevations in [Ca(2+)](i) persisted in a subset of cells even at basal glucose. Isolated β-cells showed elevated insulin secretion at basal glucose; however, insulin secretion from Cx36(-/-) islets was minimally altered. [Ca(2+)](i) was further elevated under basal conditions, but insulin release still suppressed in K(ATP) loss-of-function islets. Forced elevation of cAMP led to PKA-mediated increases in insulin secretion from islets lacking gap-junctions, but not from islets expressing Cx36 gap junctions. We conclude there is a redundancy in how cell-cell communication in the islet suppresses insulin release. Gap junctions suppress cellular heterogeneity and spontaneous [Ca(2+)](i) signals, while other juxtacrine mechanisms, regulated by PKA and glucose, suppress more distal steps in exocytosis. Each mechanism is sufficiently robust to compensate for a loss of the other and still suppress basal insulin secretion.

Elevated levels of branched-chain amino acids have little effect on pancreatic islet cells, but L-arginine impairs function through activation of the endoplasmic reticulum stress response.

PubMed

Mullooly, Niamh; Vernon, Wendy; Smith, David M; Newsholme, Philip

2014-03-01

Recent metabolic profiling studies have identified a correlation between branched-chain amino acid levels, insulin resistance associated with prediabetes and susceptibility to type 2 diabetes. Glucose and lipids in chronic excess have been reported to induce toxic effects in pancreatic β-cells, but the effect of elevated amino acid concentrations on primary islet cell function has not been investigated to date. The aim of this study was to investigate the effect of chronic exposure to various amino acids on islet cell function in vitro. Isolated rat islets were incubated over periods of 48 h with a range of concentrations of individual amino acids (0.1 μm to 10 mm). After 48 h, islets were assessed for glucose-dependent insulin secretion capacity, proliferation or islet cell apoptosis. We report that elevated levels of branched-chain amino acids have little effect on pancreatic islet cell function or viability; however, increased levels of the amino acid l-arginine were found to be β-cell toxic, causing a dose-dependent decrease in insulin secretion accompanied by a decrease in islet cell proliferation and an increase in islet cell apoptosis. These effects were not due to l-arginine-dependent increases in production of nitric oxide but arose through elicitation of the islet cell endoplasmic reticulum stress response. This novel finding indicates, for the first time, that the l-arginine concentration in vitro may impact negatively on islet cell function, thus indicating further complexity in relationship to in vivo susceptibility of β-cells to nutrient-induced dysfunction.

Different digestion enzymes used for human pancreatic islet isolation: A mixed treatment comparison (MTC) meta-analysis

PubMed Central

Rheinheimer, Jakeline; Ziegelmann, Patrícia Klarmann; Carlessi, Rodrigo; Reck, Luciana Ross; Bauer, Andrea Carla; Leitão, Cristiane Bauermann; Crispim, Daisy

2014-01-01

Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval – CrI) = −2.19 (−4.25 to −0.21) and −2.28 (−4.49 to −0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = −1.69 (−2.87 to −0.51) and −1.07 (−1.79 to −0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF. PMID:25437379

Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

NASA Astrophysics Data System (ADS)

Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

1992-09-01

The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

Importance of Extranuclear Estrogen Receptor-α and Membrane G Protein–Coupled Estrogen Receptor in Pancreatic Islet Survival

PubMed Central

Liu, Suhuan; Le May, Cedric; Wong, Winifred P.S.; Ward, Robert D.; Clegg, Deborah J.; Marcelli, Marco; Korach, Kenneth S.; Mauvais-Jarvis, Franck

2009-01-01

OBJECTIVE We showed that 17β-estradiol (E2) favors pancreatic β-cell survival via the estrogen receptor-α (ERα) in mice. E2 activates nuclear estrogen receptors via an estrogen response element (ERE). E2 also activates nongenomic signals via an extranuclear form of ERα and the G protein–coupled estrogen receptor (GPER). We studied the contribution of estrogen receptors to islet survival. RESEARCH DESIGN AND METHODS We used mice and islets deficient in estrogen receptor-α (αERKO−/−), estrogen receptor-β (βERKO−/−), estrogen receptor-α and estrogen receptor-β (αβERKO−/−), and GPER (GPERKO−/−); a mouse lacking ERα binding to the ERE; and human islets. These mice and islets were studied in combination with receptor-specific pharmacological probes. RESULTS We show that ERα protection of islet survival is ERE independent and that E2 favors islet survival through extranuclear and membrane estrogen receptor signaling. We show that ERβ plays a minor cytoprotective role compared to ERα. Accordingly, βERKO−/− mice are mildly predisposed to streptozotocin-induced islet apoptosis. However, combined elimination of ERα and ERβ in mice does not synergize to provoke islet apoptosis. In αβERKO−/− mice and their islets, E2 partially prevents apoptosis suggesting that an alternative pathway compensates for ERα/ERβ deficiency. We find that E2 protection of islet survival is reproduced by a membrane-impermeant E2 formulation and a selective GPER agonist. Accordingly, GPERKO−/− mice are susceptible to streptozotocin-induced insulin deficiency. CONCLUSIONS E2 protects β-cell survival through ERα and ERβ via ERE-independent, extra-nuclear mechanisms, as well as GPER-dependent mechanisms. The present study adds a novel dimension to estrogen biology in β-cells and identifies GPER as a target to protect islet survival. PMID:19587358

Islet Hypersensitivity to Glucose Is Associated With Disrupted Oscillations and Increased Impact of Proinflammatory Cytokines in Islets From Diabetes-Prone Male Mice

PubMed Central

Corbin, Kathryn L.; Waters, Christopher D.; Shaffer, Brett K.; Verrilli, Gretchen M.

2016-01-01

Pulsatile insulin release is the primary means of blood glucose regulation. The loss of pulsatility is thought to be an early marker and possible factor in developing type 2 diabetes. Another early adaptation in islet function to compensate for obesity is increased glucose sensitivity (left shift) associated with increased basal insulin release. We provide evidence that oscillatory disruptions may be linked with overcompensation (glucose hypersensitivity) in islets from diabetes-prone mice. We isolated islets from male 4- to 5-week-old (prediabetic) and 10- to 12-week-old (diabetic) leptin-receptor-deficient (db/db) mice and age-matched heterozygous controls. After an overnight incubation in media with 11 mM glucose, we measured islet intracellular calcium in 5, 8, 11, or 15 mM glucose. Islets from heterozygous 10- to 12-week-old mice were quiescent in 5 mM glucose and displayed oscillations with increasing amplitude and/or duration in 8, 11, and 15 mM glucose, respectively. Islets from diabetic 10- to 12-week-old mice, in contrast, showed robust oscillations in 5 mM glucose that declined with increasing glucose. Similar trends were observed at 4–5-weeks of age. A progressive left shift in maximal insulin release was also observed in islets as db/db mice aged. Reducing glucokinase activity with 1 mM D-mannoheptulose restored oscillations in 11 mM glucose. Finally, overnight low-dose cytokine exposure negatively impacted oscillations preferentially in high glucose in diabetic islets compared with heterozygous controls. Our findings suggest the following: 1) islets from frankly diabetic mice can produce oscillations, 2) elevated sensitivity to glucose prevents diabetic mouse islets from producing oscillations in normal postprandial (11–15 mM glucose) conditions, and 3) hypersensitivity to glucose may magnify stress effects from inflammation or other sources. PMID:26943366

Oxygenated thawing and rewarming alleviate rewarming injury of cryopreserved pancreatic islets.

PubMed

Komatsu, Hirotake; Barriga, Alyssa; Medrano, Leonard; Omori, Keiko; Kandeel, Fouad; Mullen, Yoko

2017-05-06

Pancreatic islet transplantation is an effective treatment for Type 1 diabetic patients to eliminate insulin injections; however, a shortage of donor organs hinders the widespread use. Although long-term islet storage, such as cryopreservation, is considered one of the key solutions, transplantation of cryopreserved islets is still not practical due to the extensive loss during the cryopreservation-rewarming process. We have previously reported that culturing islets in a hyperoxic environment is an effective treatment to prevent islet death from the hypoxic injury during culture. In this study, we explored the effectiveness of thawing and rewarming cryopreserved islets in a hyperoxic environment. Following cryopreservation of isolated human islets, the thawing solution and culture media were prepared with or without pre-equilibration to 50% oxygen. Thawing/rewarming and the pursuant two-day culture were performed with or without oxygenation. Short-term recovery rate, defined as the volume change during cryopreservation and thawing/rewarming, was assessed. Ischemia-associated and inflammation-associated gene expressions were examined using qPCR after the initial rewarming period. Long-term recovery rate, defined as the volume change during the two-day culture after the thawing/rewarming, was also examined. Islet metabolism and function were assessed by basal oxygen consumption rate and glucose stimulated insulin secretion after long-term recovery. Oxygenated thawing/rewarming did not alter the short-term recovery rate. Inflammation-associated gene expressions were elevated by the conventional thawing/rewarming method and suppressed by the oxygenated thawing/rewarming, whereas ischemia-associated gene expressions did not change between the thawing/rewarming methods. Long-term recovery rate experiments revealed that only the combination therapy of oxygenated thawing/rewarming and oxygenated culture alleviated islet volume loss. These islets showed higher metabolism and better function among the conditions examined. Oxygenated thawing/rewarming alleviated islet volume loss, with the help of oxygenated culture. Copyright © 2017. Published by Elsevier Inc.

A role of pancreatic stellate cells in islet fibrosis and β-cell dysfunction in type 2 diabetes mellitus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Esder; Ryu, Gyeong Ryul; Ko, Seung-Hyun

Objectives: To investigate whether the activation of pancreatic stellate cells (PSCs) leads to pancreatic β-cell dysfunction in type 2 diabetes mellitus (T2DM). Methods: The pancreases of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of T2DM, and patient with T2DM were analyzed. And the in vitro and in vivo effects of pirfenidone, an antifibrotic agent, on PSC activation, islet fibrosis, and β-cells were studied. Results: The extent of islet fibrosis and the percentage of activated PSCs, positive for α-smooth muscle actin, in the islets were significantly greater in OLETF rats compared with non-diabetic rats. Also, the extent of islet fibrosis inmore » patients with T2DM was slightly greater compared with age- and BMI-matched non-diabetic patients. In rat PSCs cultured with high glucose for 72 h, pirfenidone produced decreases in cell proliferation, release of collagen, and the expression of fibronectin and connective tissue growth factor. Treatment of OLETF rats with pirfenidone for 16 weeks decreased the activation of PSCs and the extent of islet fibrosis, but did not enhance glucose tolerance, pancreatic insulin content, or β-cell mass. Conclusions: Activated PSCs in islets might lead to islet fibrosis in T2DM. However, PSC activation itself might not contribute significantly to progressive β-cell failure in T2DM. - Highlights: • Islet fibrosis developed progressively in OLETF rats, a model of type 2 diabetes. • PSCs in the islets became activated in OLETF rats. • Islet fibrosis was increased in patients with type 2 diabetes. • Pirfenidone attenuated the activation of PSCs and islet fibrosis in OLETF rats. • Pirfenidonet had no effects on glucose tolerance or on β-cells in OLETF rats.« less

Effectiveness of a web-based automated cell distribution system.

PubMed

Niland, Joyce C; Stiller, Tracey; Cravens, James; Sowinski, Janice; Kaddis, John; Qian, Dajun

2010-01-01

In recent years, industries have turned to the field of operations research to help improve the efficiency of production and distribution processes. Largely absent is the application of this methodology to biological materials, such as the complex and costly procedure of human pancreas procurement and islet isolation. Pancreatic islets are used for basic science research and in a promising form of cell replacement therapy for a subset of patients afflicted with severe type 1 diabetes mellitus. Having an accurate and reliable system for cell distribution is therefore crucial. The Islet Cell Resource Center Consortium was formed in 2001 as the first and largest cooperative group of islet production and distribution facilities in the world. We previously reported on the development of a Matching Algorithm for Islet Distribution (MAID), an automated web-based tool used to optimize the distribution of human pancreatic islets by matching investigator requests to islet characteristics. This article presents an assessment of that algorithm and compares it to the manual distribution process used prior to MAID. A comparison was done using an investigator's ratio of the number of islets received divided by the number requested pre- and post-MAID. Although the supply of islets increased between the pre- versus post-MAID period, the median received-to-requested ratio remained around 60% due to an increase in demand post-MAID. A significantly smaller variation in the received-to-requested ratio was achieved in the post- versus pre-MAID period. In particular, the undesirable outcome of providing users with more islets than requested, ranging up to four times their request, was greatly reduced through the algorithm. In conclusion, this analysis demonstrates, for the first time, the effectiveness of using an automated web-based cell distribution system to facilitate efficient and consistent delivery of human pancreatic islets by enhancing the islet matching process.

Mechanisms of β-Cell Death in Response to Double-Stranded (ds) RNA and Interferon-γ

PubMed Central

Scarim, Anna L.; Arnush, Marc; Blair, Libby A.; Concepcion, Josephine; Heitmeier, Monique R.; Scheuner, Donalyn; Kaufman, Randal J.; Ryerse, Jan; Buller, R. Mark; Corbett, John A.

2001-01-01

Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate β-cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs β-cell function and induces β-cell death. The synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly IC) stimulates β-cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-γ stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC + IFN-γ on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and NG-monomethyl-l-arginine, attenuate poly IC + IFN-γ-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. NG-monomethyl-l-arginine fails to prevent poly IC- and poly IC + IFN-γ-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC- and poly IC + IFN-γ-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; however, nitric oxide-dependent DNA damage induced by the combination of poly IC + IFN-γ is not attenuated by the genetic absence of PKR. These findings indicate that dsRNA stimulates PKR-dependent islet cell apoptosis, an event that is associated with normal islet secretory function. In contrast, poly IC + IFN-γ-induced inhibition of glucose-stimulated insulin secretion and islet cell necrosis are events that are mediated by islet production of nitric oxide. These findings suggest that at least one IFN-γ-induced antiviral response (islet cell necrosis) is mediated through a PKR-independent pathway. PMID:11438474

Dual role of interleukin-1β in islet amyloid formation and its β-cell toxicity: Implications for type 2 diabetes and islet transplantation.

PubMed

Park, Yoo Jin; Warnock, Garth L; Ao, Ziliang; Safikhan, Nooshin; Meloche, Mark; Asadi, Ali; Kieffer, Timothy J; Marzban, Lucy

2017-05-01

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to β-cell failure in type 2 diabetes, cultured and transplanted islets. We previously showed that biosynthetic hIAPP aggregates induce β-cell Fas upregulation and activation of the Fas apoptotic pathway. We used cultured human and hIAPP-expressing mouse islets to investigate: (1) the role of interleukin-1β (IL-1β) in amyloid-induced Fas upregulation; and (2) the effects of IL-1β-induced β-cell dysfunction on pro-islet amyloid polypeptide (proIAPP) processing and amyloid formation. Human and h IAPP -expressing mouse islets were cultured to form amyloid without or with the IL-1 receptor antagonist (IL-1Ra) anakinra, in the presence or absence of recombinant IL-1β. Human islets in which amyloid formation was prevented (amyloid inhibitor or Ad-prohIAPP-siRNA) were cultured similarly. β-cell function, apoptosis, Fas expression, caspase-8 activation, islet IL-1β, β-cell area, β-/α-cell ratio, amyloid formation, and (pro)IAPP forms were assessed. hIAPP aggregates were found to increase IL-1β levels in cultured human islets that correlated with β-cell Fas upregulation, caspase-8 activation and apoptosis, all of which were reduced by IL-1Ra treatment or prevention of amyloid formation. Moreover, IL-1Ra improved culture-induced β-cell dysfunction and restored impaired proIAPP processing, leading to lower amyloid formation. IL-1β treatment potentiated impaired proIAPP processing and increased amyloid formation in cultured human and h IAPP -expressing mouse islets, which were prevented by IL-1Ra. IL-1β plays a dual role by: (1) mediating amyloid-induced Fas upregulation and β-cell apoptosis; (2) inducing impaired proIAPP processing thereby potentiating amyloid formation. Blocking IL-1β may provide a new strategy to preserve β cells in conditions associated with islet amyloid formation. © 2017 John Wiley & Sons Ltd.

Polymeric microsphere-facilitated site-specific delivery of quercetin prevents senescence of pancreatic islets in vivo and improves transplantation outcomes in mouse model of diabetes.

PubMed

Pathak, Shiva; Regmi, Shobha; Nguyen, Tiep Tien; Gupta, Biki; Gautam, Milan; Yong, Chul Soon; Kim, Jong Oh; Son, Youlim; Kim, Jae-Ryong; Park, Min Hui; Bae, Young Kyung; Park, So Young; Jeong, Daewon; Yook, Simmyung; Jeong, Jee-Heon

2018-06-05

Attenuation of senescence progression may be attractive way to preserve the functionality of pancreatic islets (PI) after transplantation. In this study, we developed a model for in vitro induction of premature senescence in rat PI and showed the effectiveness of quercetin (QU) to prevent the senescence. To provide targeted-delivery of QU to the PI after transplantation, we prepared the hybrid clusters (HC) of islet single cells (ISC) and QU-loaded polymeric microspheres (QU; ∼7.55 ng HC -1 ). Long-term culture of the HC revealed reduced levels of reactive oxygen species and decreased expression of senescence-associated beta galactosidase, Rb, p53, p16, and p21 compared to that of the control islets. Transplantation of HC into subcutaneous space of the immune-deficient mice produced better glycemic control compared to the control islets or the ICC-transplanted mice. SA-β-Gal staining of the in vivo transplanted HC sample showed lower intensity compared to that of the control islets or the islet cell clusters. Thus, in situ delivery of therapeutic agent may be a promising approach to improve therapeutic outcomes in cell therapy. In this study, we aimed to improve outcomes in islet transplantation using in situ delivery of quercetin to pancreatic islets, using polymeric microspheres. We prepared prolonged release-type microspheres and constructed hybrid clusters of pancreatic islets and the microspheres using hanging drop method. The presence of quercetin in the cellular microenvironment attenuated the progression of senescence in the pancreatic islets in a long-term in vitro culture. Moreover, transplantation of the hybrid clusters in the diabetic mice produced better glycemic control compared to that of the control islets. In addition, quercetin delayed the progression of senescence in the pancreatic islets after in vivo transplantation. Thus, local delivery of antioxidants like quercetin may be an attractive way to improve outcomes in cell therapy. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

PubMed

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K

2008-03-01

BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans

PubMed Central

Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K.

2010-01-01

Background A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO®13, SYTO®24 and SYBR®14 as possible alternatives to FDA. Results We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO®13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. Conclusions From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability. PMID:20814586

Metabolomics applied to the pancreatic islet.

PubMed

Gooding, Jessica R; Jensen, Mette V; Newgard, Christopher B

2016-01-01

Metabolomics, the characterization of the set of small molecules in a biological system, is advancing research in multiple areas of islet biology. Measuring a breadth of metabolites simultaneously provides a broad perspective on metabolic changes as the islets respond dynamically to metabolic fuels, hormones, or environmental stressors. As a result, metabolomics has the potential to provide new mechanistic insights into islet physiology and pathophysiology. Here we summarize advances in our understanding of islet physiology and the etiologies of type-1 and type-2 diabetes gained from metabolomics studies. Copyright © 2015 Elsevier Inc. All rights reserved.

[Progress in isolation and purification of porcine islets].

PubMed

Zhu, Haitao; Yu, Liang; Wang, Bo

2012-08-01

To review the common methods of isolation and purification of porcine islets and research progress. Domestic and abroad literature concerning the isolation and purification of porcine islets was reviewed and analyzed thoroughly. The efficacy of the isolation and purification depends on the selection of donor, the procurement and cryopreservation of high-quality donor pancreas, and the selection and improvement of the operation. The shortage of transplanted islets could be resolved by the establishment of standardized and optimal process, which may also promote the development of porcine islet xenograft.

Islet grafting and imaging in a bioengineered intramuscular space†

PubMed Central

Witkowski, Piotr; Sondermeijer, Hugo; Hardy, Mark A.; Woodland, David C.; Lee, Keagan; Bhagat, Govind; Witkowski, Kajetan; See, Fiona; Rana, Abbas; Maffei, Antonella; Itescu, Silviu; Harris, Paul E.

2011-01-01

Background Since the hepatic portal system may not be the optimal site for islet transplantation, several extrahepatic sites have been studied. Here we examine an intramuscular transplantation site, bioengineered to better support islet neovascularization, engraftment, and survival, and demonstrate that at this novel site, grafted beta cell mass may be quantitated in a real time non-invasive manner by PET imaging. Methods Streptozotocin induced rats were pretreated intramuscularly with a biocompatible angiogenic scaffold received syngeneic islet transplants 2 weeks later. The recipients were monitored serially by blood glucose and glucose tolerance measurements and by PET imaging of the transplant site with [11C] dihydrotetrabenazine. Parallel histopathologic evaluation of the grafts was done using insulin staining and evaluation of microvasularity. Results Reversal of hyperglycemia by islet transplantation was most successful in recipients pretreated with bioscaffolds containing angiogenic factors as compared to those who received no bioscaffolds or bioscaffolds not treated with angiogenic factors. PET imaging with [11C] dihydrotetrabenazine, insulin staining and microvascular density patterns were consistent with islet survival, increased levels of angiogenesis, and with reversal of hyperglycemia. Conclusions Induction of increased neovascularization at an intramuscular site significantly improves islet transplant engraftment and survival compared to controls. The use of a non hepatic transplant site may avoid intrahepatic complications and permit the use of PET imaging to measure and follow transplanted beta-cell mass in real time. These findings have important implications for effective islet implantation outside of the liver, and offer promising possibilities for improving islet survival, monitoring, and even prevention of islet loss. PMID:19898201

Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

PubMed

Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

2016-10-01

The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

Anti-Inflammatory Strategies in Intrahepatic Islet Transplantation: A Comparative Study in Preclinical Models.

PubMed

Citro, Antonio; Cantarelli, Elisa; Pellegrini, Silvia; Dugnani, Erica; Piemonti, Lorenzo

2018-02-01

The identification of pathway(s) playing a pivotal role in peritransplant detrimental inflammatory events represents the crucial step toward a better management and outcome of pancreatic islet transplanted patients. Recently, we selected the CXCR1/2 inhibition as a relevant strategy in enhancing pancreatic islet survival after transplantation. Here, the most clinically used anti-inflammatory compounds (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with a CXCR1/2 inhibitor were evaluated in their ability to improve engraftment or delay graft rejection. To rule out bias related to transplantation site, we used well-established preclinical syngeneic (250 C57BL/6 equivalent islets in C57BL/6) and allogeneic (400 Balb/c equivalent islets in C57BL6) intrahepatic islet transplantation platforms. In mice, we confirmed that targeting the CXCR1/2 pathway is crucial in preserving islet function and improving engraftment. In the allogeneic setting, CXCR1/2 inhibitor alone could reduce the overall recruitment of transplant-induced leukocytes and significantly prolong the time to graft rejection both as a single agent and in combination with immunosuppression. No other anti-inflammatory compounds tested (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with CXCR1/2 inhibitor improve islet engraftment and significantly delay graft rejection in the presence of MMF + FK-506 immunosuppressive treatment. These findings indicate that only the CXCR1/2-mediated axis plays a crucial role in controlling the islet damage and should be a target for intervention to improve the efficiency of islet transplantation.

In vivo imaging of autologous islet grafts in the liver and under the kidney capsule in non-human primates

PubMed Central

Medarova, Zdravka; Vallabhajosyula, Prashanth; Tena, Aseda; Evgenov, Natalia; Pantazopoulos, Pamela; Tchipashvili, Vaja; Weir, Gordon; Sachs, David; Moore, Anna

2009-01-01

Objective As islet transplantation begins to show promise clinically, there is a critical need for reliable, non-invasive techniques to monitor islet graft survival. Previous work in our laboratory has shown that human islets labeled with a superparamagnetic iron oxide contrast agent and transplanted into mice could be detected by magnetic resonance imaging (MRI). The potential translation of these findings to the clinical situation requires validation of our methodology in a non-human primate model, which we have now carried out in baboons (Papio hamadryas) and reported here. Research Design and Methods: For islet labeling, we adapted the FDA-approved superparamagnetic iron oxide contrast agent, Feridex, which is used clinically for liver imaging. After partial pancreatectomy, Feridex-labeled islets were prepared and autotransplanted underneath the renal capsule and into the liver. Longitudinal in vivo MRI at days 1, 3, 8, 16, 23, and 30 after transplantation was performed in order to track the islet grafts. Results The renal subcapsular islet graft was easily detectable on T2*-weighted MRI images as a pocket of signal loss disrupting the contour of the kidney at the transplantation site. Islets transplanted in the liver appeared as distinct signal voids dispersed throughout the liver parenchyma. A semi-automated computational analysis of our MR imaging data established the feasibility of monitoring both the renal and intrahepatic grafts during the studied post-transplantation period. Conclusion This study establishes a method for the noninvasive, longitudinal detection of pancreatic islets transplanted into non-human primates using a low field clinical MRI system. PMID:19502957

Voluntary running exercise prevents β-cell failure in susceptible islets of the Zucker diabetic fatty rat.

PubMed

Delghingaro-Augusto, Viviane; Décary, Simon; Peyot, Marie-Line; Latour, Martin G; Lamontagne, Julien; Paradis-Isler, Nicolas; Lacharité-Lemieux, Marianne; Akakpo, Huguette; Birot, Olivier; Nolan, Christopher J; Prentki, Marc; Bergeron, Raynald

2012-01-15

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving β-cell function is uncertain. We evaluated the role of physical activity on β-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with β-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key β-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining β-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and β-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered β-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.

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

PubMed

Schive, Simen W; Foss, Aksel; Sahraoui, Afaf; Kloster-Jensen, Kristine; Hafsahl, Geir; Kvalheim, Gunnar; Lundgren, Torbjørn; von Zur-Mühlen, Bengt; Felldin, Marie; Rafael, Ehab; Lempinen, Marko; Korsgren, Olle; Jenssen, Trond G; Mishra, Vinod; Scholz, Hanne

2017-01-01

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 4 years after their last transplantation. An estimated 70% maintain at least partial graft function, defined as fasting C-peptide >0.1 nmol L -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 4 years after the initial islet transplantation. The average cost for an islet transplantation procedure was 347 297±60 588 NOK, or 35 424±6182 EUR, 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. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Microfluidic glucose stimulation reveals limited coordination of intracellular Ca2+ activity oscillations in pancreatic islets

PubMed Central

Rocheleau, Jonathan V.; Walker, Glenn M.; Head, W. Steven; McGuinness, Owen P.; Piston, David W.

2004-01-01

The pancreatic islet is a functional microorgan involved in maintaining normoglycemia through regulated secretion of insulin and other hormones. Extracellular glucose stimulates insulin secretion from islet β cells through an increase in redox state, which can be measured by NAD(P)H autofluorescence. Glucose concentrations over ≈7 mM generate synchronous oscillations in β cell intracellular Ca2+ concentration ([Ca2+]i), which lead to pulsatile insulin secretion. Prevailing models assume that the pancreatic islet acts as a functional syncytium, and the whole islet [Ca2+]i response has been modeled in terms of islet bursting and pacemaker models. To test these models, we developed a microfluidic device capable of partially stimulating an islet, while allowing observation of the NAD(P)H and [Ca2+]i responses. We show that β cell [Ca2+]i oscillations occur only within regions stimulated with more than ≈6.6 mM glucose. Furthermore, we show that tolbutamide, an antagonist of the ATP-sensitive K+ channel, allows these oscillations to travel farther into the nonstimulated regions of the islet. Our approach shows that the extent of Ca2+ propagation across the islet depends on a delicate interaction between the degree of coupling and the extent of ATP-sensitive K+-channel activation and illustrates an experimental paradigm that will have utility for many other biological systems. PMID:15317941

Prediction of Marginal Mass Required for Successful Islet Transplantation

PubMed Central

Papas, Klearchos K.; Colton, Clark K.; Qipo, Andi; Wu, Haiyan; Nelson, Rebecca A.; Hering, Bernhard J.; Weir, Gordon C.; Koulmanda, Maria

2013-01-01

Islet quality assessment methods for predicting diabetes reversal (DR) following transplantation are needed. We investigated two islet parameters, oxygen consumption rate (OCR) and OCR per DNA content, to predict transplantation outcome and explored the impact of islet quality on marginal islet mass for DR. Outcomes in immunosuppressed diabetic mice were evaluated by transplanting mixtures of healthy and purposely damaged rat islets for systematic variation of OCR/DNA over a wide range. The probability of DR increased with increasing transplanted OCR and OCR/DNA. On coordinates of OCR versus OCR/DNA, data fell into regions in which DR occurred in all, some, or none of the animals with a sharp threshold of around 150-nmol/min mg DNA. A model incorporating both parameters predicted transplantation outcome with sensitivity and specificity of 93% and 94%, respectively. Marginal mass was not constant, depended on OCR/DNA, and increased from 2,800 to over 100,000 islet equivalents/kg body weight as OCR/DNA decreased. We conclude that measurements of OCR and OCR/DNA are useful for predicting transplantation outcome in this model system, and OCR/DNA can be used to estimate the marginal mass required for reversing diabetes. Because human clinical islet preparations in a previous study had OCR/DNA values in the range of 100–150-nmol/min mg DNA, our findings suggest that substantial improvement in transplantation outcome may accompany increasedOCR/DNAin clinical islet preparations. PMID:20233002

Clinical islet isolation and transplantation outcomes with deceased cardiac death donors are similar to neurological determination of death donors.

PubMed

Andres, Axel; Kin, Tatsuya; O'Gorman, Doug; Livingstone, Scott; Bigam, David; Kneteman, Norman; Senior, Peter; Shapiro, A M James

2016-01-01

In islet transplantation, deceased cardiac death (DCD) donation has been identified as a potential extended source. There are currently no studies comparing outcomes between these categories, and our goal was to compare islet isolation success rates and transplantation outcomes between DCD and neurological determination of death (NDD) donors. Islet isolations from 15 DCD and 418 NDD were performed in our centre between September 2008 and September 2014. Donor variables, islet yields, metabolic function of isolated isled and insulin requirements at 1-month post-transplant were compared. Compared to NDD, pancreata from DCD were more often procured locally and donors required less vasopressive support (P < 0.001 and P = 0.023, respectively), but the other variables were similar between groups. Pre- and postpurification islet yields were similar between NDD and DCD (576 vs. 608 × 10(3) islet equivalent, P = 0.628 and 386 vs. 379, P = 0.881, respectively). The metabolic function was similar between NDD and DCD, as well as the mean decrease in insulin requirement at 1-month post-transplantation (NDD: 64.82%; DCD: 60.17% reduction, P = 0.517). These results support the broader use of DCD pancreata for islet isolation. A much larger DCD islet experience will be required to truly determine noninferiority of both short- and long-term outcomes. © 2015 Steunstichting ESOT.

Pancreatic islet isolation variables in non-human primates (rhesus macaques).

PubMed

Andrades, P; Asiedu, C K; Gansuvd, B; Inusah, S; Goodwin, K J; Deckard, L A; Jargal, U; Thomas, J M

2008-07-01

Non-human primates (NHPs) are important preclinical models for pancreatic islet transplantation (PIT) because of their close phylogenetic and immunological relationship with humans. However, low availability of NHP tissue, long learning curves and prohibitive expenses constrain the consistency of isolated NHP islets for PIT studies. To advance preclinical studies, we attempted to identify key variables that consistently influence the quantity and quality of NHP islets. Seventy-two consecutive pancreatic islet isolations from rhesus macaques were reviewed retrospectively. A scaled down, semi-automated islet isolation method was used, and monkeys with streptozotocin-induced diabetes, weighing 3-7 kg, served as recipients for allotransplantation. We analysed the effects of 22 independent variables grouped as donor factors, surgical factors and isolation technique factors. Islet yields, success of isolation and transplantation results were used as quantitative and qualitative outcomes. In the multivariate analysis, variables that significantly affected islet yield were the type of monkey, pancreas preservation, enzyme lot and volume of enzyme delivered. The variables associated with successful isolation were the enzyme lot and volume delivered. The transplant result was correlated with pancreas preservation, enzyme lot, endotoxin levels and COBE collection method. Islet quantity and quality are highly variable between isolations. The data reviewed suggest that future NHP isolations should use bilayer preservation, infuse more than 80 ml of Liberase into the pancreas, collect non-fractioned tissue from the COBE, and strictly monitor for infection.

Preoperative Computerized Tomography and Magnetic Resonance Imaging of the Pancreas Predicts Pancreatic Mass and Functional Outcomes After Total Pancreatectomy and Islet Autotransplant

PubMed Central

Young, Michael C.; Theis, Jake R.; Hodges, James S.; Dunn, Ty B.; Pruett, Timothy L.; Chinnakotla, Srinath; Walker, Sidney P.; Freeman, Martin L.; Trikudanathan, Guru; Arain, Mustafa; Robertson, R. Paul; Wilhelm, Joshua J.; Schwarzenberg, Sarah J.; Bland, Barbara; Beilman, Gregory J.; Bellin, Melena D.

2015-01-01

Objectives About two-thirds of patients will remain on insulin therapy after total pancreatectomy with islet autotransplant (TPIAT) for chronic pancreatitis. We investigated the relationship between measured pancreas volume on computerized tomography (CT) or magnetic resonance imaging (MRI), and features of chronic pancreatiits on imaging, with subsequent islet isolation and diabetes outcomes. Methods CT or MRI was reviewed for pancreas volume (Vitrea software), and presence or absence of calcifications, atrophy, and dilated pancreatic duct in 97 patients undergoing TPIAT. Relationship between these features and: (1) islet mass isolated and (2) diabetes status at 1 year post-TPAIT were evaluated. Results Pancreas volume correlated with islet mass measured as total islet equivalents (r=0.50, p<0.0001). Mean islet equivalents was reduced by more than half if any one of calcifications, atrophy, or ductal dilatation were observed. Pancreatic calcifications increased the odds of insulin dependence 4.0 fold (1.1, 15). Collectively, the pancreas volume and 3 imaging features strongly associated with 1 year insulin use (p=0.07), islet graft failure (p=0.003), Hemoglobin A1c (p=0.0004), fasting glucose (p=0.027), and fasting C-peptide level (p=0.008). Conclusions Measures of pancreatic parenchymal destruction on imaging, including smaller pancreas volume and calcifications associate strongly with impaired islet mass and 1 year diabetes outcomes. PMID:26745861

Preoperative Computerized Tomography and Magnetic Resonance Imaging of the Pancreas Predicts Pancreatic Mass and Functional Outcomes After Total Pancreatectomy and Islet Autotransplant.

PubMed

Young, Michael C; Theis, Jake R; Hodges, James S; Dunn, Ty B; Pruett, Timothy L; Chinnakotla, Srinath; Walker, Sidney P; Freeman, Martin L; Trikudanathan, Guru; Arain, Mustafa; Robertson, Paul R; Wilhelm, Joshua J; Schwarzenberg, Sarah J; Bland, Barbara; Beilman, Gregory J; Bellin, Melena D

2016-08-01

Approximately two thirds of patients will remain on insulin therapy after total pancreatectomy with islet autotransplant (TPIAT) for chronic pancreatitis. We investigated the relationship between measured pancreas volume on computerized tomography or magnetic resonance imaging and features of chronic pancreatitis on imaging, with subsequent islet isolation and diabetes outcomes. Computerized tomography or magnetic resonance imaging was reviewed for pancreas volume (Vitrea software) and presence or absence of calcifications, atrophy, and dilated pancreatic duct in 97 patients undergoing TPIAT. Relationship between these features and (1) islet mass isolated and (2) diabetes status at 1-year post-TPIAT were evaluated. Pancreas volume correlated with islet mass measured as total islet equivalents (r = 0.50, P < 0.0001). Mean islet equivalents were reduced by more than half if any one of calcifications, atrophy, or ductal dilatation were observed. Pancreatic calcifications increased the odds of insulin dependence 4.0 fold (1.1, 15). Collectively, the pancreas volume and 3 imaging features strongly associated with 1-year insulin use (P = 0.07), islet graft failure (P = 0.003), hemoglobin A1c (P = 0.0004), fasting glucose (P = 0.027), and fasting C-peptide level (P = 0.008). Measures of pancreatic parenchymal destruction on imaging, including smaller pancreas volume and calcifications, associate strongly with impaired islet mass and 1-year diabetes outcomes.

Hyaluronan and Hyaluronan-Binding Proteins Accumulate in Both Human Type 1 Diabetic Islets and Lymphoid Tissues and Associate With Inflammatory Cells in Insulitis

PubMed Central

Bogdani, Marika; Johnson, Pamela Y.; Potter-Perigo, Susan; Nagy, Nadine; Day, Anthony J.; Bollyky, Paul L.

2014-01-01

Hyaluronan (HA) is an extracellular matrix glycosaminoglycan that is present in pancreatic islets, but little is known about its involvement in the development of human type 1 diabetes (T1D). We have evaluated whether pancreatic islets and lymphoid tissues of T1D and nondiabetic organ donors differ in the amount and distribution of HA and HA-binding proteins (hyaladherins), such as inter-α-inhibitor (IαI), versican, and tumor necrosis factor–stimulated gene-6 (TSG-6). HA was dramatically increased both within the islet and outside the islet endocrine cells, juxtaposed to islet microvessels in T1D. In addition, HA was prominent surrounding immune cells in areas of insulitis. IαI and versican were present in HA-rich areas of islets, and both molecules accumulated in diabetic islets and regions exhibiting insulitis. TSG-6 was observed within the islet endocrine cells and in inflammatory infiltrates. These patterns were only observed in tissues from younger donors with disease duration of <10 years. Furthermore, HA and IαI amassed in follicular germinal centers and in T-cell areas in lymph nodes and spleens in T1D patients compared with control subjects. Our observations highlight potential roles for HA and hyaladherins in the pathogenesis of diabetes. PMID:24677718

Transgenic mice overexpressing insulin-like growth factor-II in β cells develop type 2 diabetes

PubMed Central

Devedjian, Jean-Christophe; George, Monica; Casellas, Alba; Pujol, Anna; Visa, Joana; Pelegrín, Mireia; Gros, Laurent; Bosch, Fatima

2000-01-01

During embryonic development, insulin-like growth factor-II (IGF-II) participates in the regulation of islet growth and differentiation. We generated transgenic mice (C57BL6/SJL) expressing IGF-II in β cells under control of the rat Insulin I promoter in order to study the role of islet hyperplasia and hyperinsulinemia in the development of type 2 diabetes. In contrast to islets from control mice, islets from transgenic mice displayed high levels of IGF-II mRNA and protein. Pancreases from transgenic mice showed an increase in β-cell mass (about 3-fold) and in insulin mRNA levels. However, the organization of cells within transgenic islets was disrupted, with glucagon-producing cells randomly distributed throughout the core. We also observed enhanced glucose-stimulated insulin secretion and glucose utilization in islets from transgenic mice. These mice displayed hyperinsulinemia, mild hyperglycemia, and altered glucose and insulin tolerance tests, and about 30% of these animals developed overt diabetes when fed a high-fat diet. Furthermore, transgenic mice obtained from the N1 backcross to C57KsJ mice showed high islet hyperplasia and insulin resistance, but they also developed fatty liver and obesity. These results indicate that local overexpression of IGF-II in islets might lead to type 2 diabetes and that islet hyperplasia and hypersecretion of insulin might occur early in the pathogenesis of this disease. PMID:10727441

Simplified method to isolate highly pure canine pancreatic islets.

PubMed

Woolcott, Orison O; Bergman, Richard N; Richey, Joyce M; Kirkman, Erlinda L; Harrison, L Nicole; Ionut, Viorica; Lottati, Maya; Zheng, Dan; Hsu, Isabel R; Stefanovski, Darko; Kabir, Morvarid; Kim, Stella P; Catalano, Karyn J; Chiu, Jenny D; Chow, Robert H

2012-01-01

The canine model has been used extensively to improve the human pancreatic islet isolation technique. At the functional level, dog islets show high similarity to human islets and thus can be a helpful tool for islet research. We describe and compare 2 manual isolation methods, M1 (initial) and M2 (modified), and analyze the variables associated with the outcomes, including islet yield, purity, and glucose-stimulated insulin secretion (GSIS). Male mongrel dogs were used in the study. M2 (n = 7) included higher collagenase concentration, shorter digestion time, faster shaking speed, colder purification temperature, and higher differential density gradient than M1 (n = 7). Islet yield was similar between methods (3111.0 ± 309.1 and 3155.8 ± 644.5 islets/g, M1 and M2, respectively; P = 0.951). Pancreas weight and purity together were directly associated with the yield (adjusted R(2) = 0.61; P = 0.002). Purity was considerably improved with M2 (96.7% ± 1.2% vs 75.0% ± 6.3%; P = 0.006). M2 improved GSIS (P = 0.021). Independently, digestion time was inversely associated with GSIS. We describe an isolation method (M2) to obtain a highly pure yield of dog islets with adequate β-cell glucose responsiveness. The isolation variables associated with the outcomes in our canine model confirm previous reports in other species, including humans.

Simplified Method to Isolate Highly Pure Canine Pancreatic Islets

PubMed Central

Woolcott, Orison O.; Bergman, Richard N.; Richey, Joyce M.; Kirkman, Erlinda L.; Harrison, L. Nicole; Ionut, Viorica; Lottati, Maya; Zheng, Dan; Hsu, Isabel R.; Stefanovski, Darko; Kabir, Morvarid; Kim, Stella P.; Catalano, Karyn J.; Chiu, Jenny D.; Chow, Robert H.

2015-01-01

Objectives The canine model has been used extensively to improve the human pancreatic islet isolation technique. At the functional level, dog islets show high similarity to human islets and thus can be a helpful tool for islet research. We describe and compare 2 manual isolation methods, M1 (initial) and M2 (modified), and analyze the variables associated with the outcomes, including islet yield, purity, and glucose-stimulated insulin secretion (GSIS). Methods Male mongrel dogs were used in the study. M2 (n = 7) included higher collagenase concentration, shorter digestion time, faster shaking speed, colder purification temperature, and higher differential density gradient than M1 (n = 7). Results Islet yield was similar between methods (3111.0 ± 309.1 and 3155.8 ± 644.5 islets/g, M1 and M2, respectively; P = 0.951). Pancreas weight and purity together were directly associated with the yield (adjusted R2 = 0.61; P = 0.002). Purity was considerably improved with M2 (96.7% ± 1.2% vs 75.0% ± 6.3%; P = 0.006). M2 improved GSIS (P = 0.021). Independently, digestion time was inversely associated with GSIS. Conclusions We describe an isolation method (M2) to obtain a highly pure yield of dog islets with adequate β-cell glucose responsiveness. The isolation variables associated with the outcomes in our canine model confirm previous reports in other species, including humans. PMID:21792087

A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers.

PubMed

Vivot, Kevin; Benahmed, Malika A; Seyfritz, Elodie; Bietiger, William; Elbayed, Karim; Ruhland, Elisa; Langlois, Allan; Maillard, Elisa; Pinget, Michel; Jeandidier, Nathalie; Gies, Jean-Pierre; Namer, Izzie-Jacques; Sigrist, Séverine; Reix, Nathalie

2016-01-01

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1 H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1 H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation.

A Metabolomic Approach (1H HRMAS NMR Spectroscopy) Supported by Histology to Study Early Post-transplantation Responses in Islet-transplanted Livers

PubMed Central

Vivot, Kevin; Benahmed, Malika A.; Seyfritz, Elodie; Bietiger, William; Elbayed, Karim; Ruhland, Elisa; Langlois, Allan; Maillard, Elisa; Pinget, Michel; Jeandidier, Nathalie; Gies, Jean-Pierre; Namer, Izzie-Jacques; Sigrist, Séverine; Reix, Nathalie

2016-01-01

Intrahepatic transplantation of islets requires a lot of islets because more than 50% of the graft is lost during the 24 hours following transplantation. We analyzed, in a rat model, early post-transplantation inflammation using systemic inflammatory markers, or directly in islet-transplanted livers by immunohistochemistry. 1H HRMAS NMR was employed to investigate metabolic responses associated with the transplantation. Inflammatory markers (Interleukin-6, α2-macroglobulin) are not suitable to follow islet reactions as they are not islet specific. To study islet specific inflammatory events, immunohistochemistry was performed on sections of islet transplanted livers for thrombin (indicator of the instant blood-mediated inflammatory reaction (IBMIR)) and granulocytes and macrophages. We observed a specific correlation between IBMIR and granulocyte and macrophage infiltration after 12 h. In parallel, we identified a metabolic response associated with transplantation: after 12 h, glucose, alanine, aspartate, glutamate and glutathione were significantly increased. An increase of glucose is a marker of tissue degradation, and could be explained by immune cell infiltration. Alanine, aspartate and glutamate are inter-connected in a common metabolic pathway known to be activated during hypoxia. An increase of glutathione revealed the presence of antioxidant protection. In this study, IBMIR visualization combined with 1H HRMAS NMR facilitated the characterization of cellular and molecular pathways recruited following islet transplantation. PMID:27766032

Poly D,L-lactide-co-glycolide (PLGA) nanoparticle-encapsulated honeybee (Apis melifera) venom promotes clearance of Salmonella enterica serovar Typhimurium infection in experimentally challenged pigs through the up-regulation of T helper type 1 specific immune responses.

PubMed

Lee, Jin-A; Jung, Bock-Gie; Kim, Tae-Hoon; Kim, Yun-Mi; Park, Min-Ho; Hyun, Pung-mi; Jeon, Jong-woon; Park, Jin-kyu; Cho, Cheong-Weon; Suh, Guk-Hyun; Lee, Bong-Joo

2014-10-15

Honeybee (Apis melifera) venom (HBV), which includes melittin and lipid-soluble ingredients (chrysin and pinocembrin), elicited increases in the CD4(+)/CD8(+) T lymphocyte ratio, relative mRNA expression levels of the T helper type 1 (Th 1) cytokines (interferon-γ and IL-12) and reinforced viral clearance of an experimental porcine reproductive and respiratory syndrome (PRRS) virus infection in our previous study. On the basis of that previous study, we have now developed poly-d,l-lactide-co-glycolide (PLGA)-encapsulated HBV nanoparticles (P-HBV) for longer sustained release of HBV. We administered P-HBV to pigs via the rectal route, and then evaluated the potential immune-enhancing and bacterial clearance effects of P-HBV against Salmonella enterica serovar Typhimurium. The CD4(+)/CD8(+) lymphocyte ratio, proliferative capacity of peripheral blood lymphocytes and relative mRNA expression levels of IFN-γ and IL-12 (produced mainly by Th1 lymphocytes) were significantly increased in the P-HBV group up to 2 weeks post-administration of P-HBV. After S. Typhimurium infection, the P-HBV group showed a marked reduction in microbial burden in feces and all tissue samples (including the ileum, cecum, colon, and mesenteric lymph node (MLN)), a significant increase in Th 1 cytokines (IFN-γ, IL-2, and IL-12) and a marked decrease in a Th 2 cytokine (IL-4) in all tissue samples and peripheral blood lymphocytes. Thus, P-HBV may be a promising strategy for immune enhancement and prevention of S. Typhimurium or other bacterial infections. Copyright © 2014 Elsevier B.V. All rights reserved.

In vivo imaging of emerging endocrine cells reveals a requirement for PI3K-regulated motility in pancreatic islet morphogenesis

PubMed Central

Freudenblum, Julia; Iglesias, José A.; Hermann, Martin; Walsen, Tanja; Wilfinger, Armin; Meyer, Dirk

2018-01-01

ABSTRACT The three-dimensional architecture of the pancreatic islet is integral to beta cell function, but the process of islet formation remains poorly understood due to the difficulties of imaging internal organs with cellular resolution. Within transparent zebrafish larvae, the developing pancreas is relatively superficial and thus amenable to live imaging approaches. We performed in vivo time-lapse and longitudinal imaging studies to follow islet development, visualizing both naturally occurring islet cells and cells arising with an accelerated timecourse following an induction approach. These studies revealed previously unappreciated fine dynamic protrusions projecting between neighboring and distant endocrine cells. Using pharmacological compound and toxin interference approaches, and single-cell analysis of morphology and cell dynamics, we determined that endocrine cell motility is regulated by phosphoinositide 3-kinase (PI3K) and G-protein-coupled receptor (GPCR) signaling. Linking cell dynamics to islet formation, perturbation of protrusion formation disrupted endocrine cell coalescence, and correlated with decreased islet cell differentiation. These studies identified novel cell behaviors contributing to islet morphogenesis, and suggest a model in which dynamic exploratory filopodia establish cell-cell contacts that subsequently promote cell clustering. PMID:29386244

Control of Insulin Secretion by Cholinergic Signaling in the Human Pancreatic Islet

PubMed Central

Molina, Judith; Rodriguez-Diaz, Rayner; Fachado, Alberto; Jacques-Silva, M. Caroline

2014-01-01

Acetylcholine regulates hormone secretion from the pancreatic islet and is thus crucial for glucose homeostasis. Little is known, however, about acetylcholine (cholinergic) signaling in the human islet. We recently reported that in the human islet, acetylcholine is primarily a paracrine signal released from α-cells rather than primarily a neural signal as in rodent islets. In this study, we demonstrate that the effects acetylcholine produces in the human islet are different and more complex than expected from studies conducted on cell lines and rodent islets. We found that endogenous acetylcholine not only stimulates the insulin-secreting β-cell via the muscarinic acetylcholine receptors M3 and M5, but also the somatostatin-secreting δ-cell via M1 receptors. Because somatostatin is a strong inhibitor of insulin secretion, we hypothesized that cholinergic input to the δ-cell indirectly regulates β-cell function. Indeed, when all muscarinic signaling was blocked, somatostatin secretion decreased and insulin secretion unexpectedly increased, suggesting a reduced inhibitory input to β-cells. Endogenous cholinergic signaling therefore provides direct stimulatory and indirect inhibitory input to β-cells to regulate insulin secretion from the human islet. PMID:24658304

Alloantibody and Autoantibody Monitoring Predicts Islet Transplantation Outcome in Human Type 1 Diabetes

PubMed Central

Piemonti, Lorenzo; Everly, Matthew J.; Maffi, Paola; Scavini, Marina; Poli, Francesca; Nano, Rita; Cardillo, Massimo; Melzi, Raffaella; Mercalli, Alessia; Sordi, Valeria; Lampasona, Vito; Espadas de Arias, Alejandro; Scalamogna, Mario; Bosi, Emanuele; Bonifacio, Ezio; Secchi, Antonio; Terasaki, Paul I.

2013-01-01

Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes. PMID:23274902

Influence of reaction medium during synthesis of Gantrez AN 119 nanoparticles for oral vaccination.

PubMed

Vandamme, Katrien; Melkebeek, Vesna; Cox, Eric; Deforce, Dieter; Lenoir, Joke; Adriaens, Els; Vervaet, Chris; Remon, Jean Paul

2010-02-01

Two synthesis methods of poly(methyl vinyl ether-co-maleic anhydride) (Gantrez AN 119) nanoparticles (NP) (used for oral vaccination) were compared. Wheat germ agglutinin (WGA) was used as ligand to enhance the bioadhesive properties of NP and beta-galactosidase as antigen. The first method encapsulated beta-galactosidase in NP by co-precipitation in an acetone/water mixture containing 44% acetone. In the second method, antigen addition occurred in 100% acetone. To improve stability, NP were crosslinked with 1,3-diaminopropane. The stability of WGA-conjugated NP with encapsulated antigen diminished at lower pH and when decreasing the amount of crosslinker. The binding type between WGA and polymer depended on the synthesis method: predominantly ionic bonds were formed using the 44% acetone method, whereas synthesis via the 100% acetone method resulted in covalent bonds. The biological activity of the WGA coating, evaluated via a pig gastric mucin binding test, was lower in NP prepared via the 100% acetone method. No release of native antigen was detected after hydrolysis of NP, due to the covalent antigen binding during antigen encapsulation and the high reactivity of the polymer. Moreover, the mucosal irritation capacity was evaluated upon nanoparticle hydrolysis using a slug mucosal irritation assay. Herein, hydrolysed NP of the 44% acetone method were classified as mild irritative. Copyright (c) 2009 Elsevier B.V. All rights reserved.

Biotin enhances ATP synthesis in pancreatic islets of the rat, resulting in reinforcement of glucose-induced insulin secretion.

PubMed

Sone, Hideyuki; Sasaki, Yuka; Komai, Michio; Toyomizu, Masaaki; Kagawa, Yasuo; Furukawa, Yuji

2004-02-13

Previous studies showed that biotin enhanced glucose-induced insulin secretion. Changes in the cytosolic ATP/ADP ratio in the pancreatic islets participate in the regulation of insulin secretion by glucose. In the present study we investigated whether biotin regulates the cytosolic ATP/ADP ratio in glucose-stimulated islets. When islets were stimulated with glucose plus biotin, the ATP/ADP ratio increased to approximately 160% of the ATP/ADP ratio in islets stimulated with glucose alone. The rate of glucose oxidation, assessed by CO(2) production, was also about 2-fold higher in islets treated with biotin. These increasing effects of biotin were proportional to the effects seen in insulin secretion. There are no previous reports of vitamins, such as biotin, directly affecting ATP synthesis. Our data indicate that biotin enhances ATP synthesis in islets following the increased rate of substrate oxidation in mitochondria and that, as a consequence of these events, glucose-induced insulin release is reinforced by biotin.

Is islet transplantation a realistic approach to curing diabetes?

PubMed

Jin, Sang-Man; Kim, Kwang-Won

2017-01-01

Since the report of type 1 diabetes reversal in seven consecutive patients by the Edmonton protocol in 2000, pancreatic islet transplantation has been reappraised based on accumulated clinical evidence. Although initially expected to therapeutically target long-term insulin independence, islet transplantation is now indicated for more specific clinical benefits. With the long-awaited report of the first phase 3 clinical trial in 2016, allogeneic islet transplantation is now transitioning from an experimental to a proven therapy for type 1 diabetes with problematic hypoglycemia. Islet autotransplantation has already been therapeutically proven in chronic pancreatitis with severe abdominal pain refractory to conventional treatments, and it holds promise for preventing diabetes after partial pancreatectomy due to benign pancreatic tumors. Based on current evidence, this review focuses on islet transplantation as a realistic approach to treating diabetes.

What Are Islet Cells?

MedlinePlus

... to put the cells What is Islet Transplantation? Sustainability - Tackling the immune system Supply - Creating more cells ... to put the cells What is Islet Transplantation? Sustainability - Tackling the immune system Supply - Creating more cells ...

Regulation of xenogeneic porcine pancreatic islets.

PubMed

Arcidiacono, Judith A; Evdokimov, Evgenij; Lee, Mark H; Jones, Jeff; Rudenko, Larisa; Schneider, Bruce; Snoy, Phillip J; Wei, Cheng-Hong; Wensky, Allen K; Wonnacott, Keith

2010-01-01

The use of xenogeneic porcine pancreatic islets has been shown to be a potentially promising alternative to using human allogeneic islets to treat insulin-dependent type 1 diabetes (T1D). This article provides an overview of the existing FDA regulatory framework that would be applied to the regulation of clinical trials utilizing xenogeneic porcine pancreatic islets to treat T1D. © 2010 John Wiley & Sons A/S.

Polymers in cell encapsulation from an enveloped cell perspective.

PubMed

de Vos, Paul; Lazarjani, Hamideh Aghajani; Poncelet, Denis; Faas, Marijke M

2014-04-01

In the past two decades, many polymers have been proposed for producing immunoprotective capsules. Examples include the natural polymers alginate, agarose, chitosan, cellulose, collagen, and xanthan and synthetic polymers poly(ethylene glycol), polyvinyl alcohol, polyurethane, poly(ether-sulfone), polypropylene, sodium polystyrene sulfate, and polyacrylate poly(acrylonitrile-sodium methallylsulfonate). The biocompatibility of these polymers is discussed in terms of tissue responses in both the host and matrix to accommodate the functional survival of the cells. Cells should grow and function in the polymer network as adequately as in their natural environment. This is critical when therapeutic cells from scarce cadaveric donors are considered, such as pancreatic islets. Additionally, the cell mass in capsules is discussed from the perspective of emerging new insights into the release of so-called danger-associated molecular pattern molecules by clumps of necrotic therapeutic cells. We conclude that despite two decades of intensive research, drawing conclusions about which polymer is most adequate for clinical application is still difficult. This is because of the lack of documentation on critical information, such as the composition of the polymer, the presence or absence of confounding factors that induce immune responses, toxicity to enveloped cells, and the permeability of the polymer network. Only alginate has been studied extensively and currently qualifies for application. This review also discusses critical issues that are not directly related to polymers and are not discussed in the other reviews in this issue, such as the functional performance of encapsulated cells in vivo. Physiological endocrine responses may indeed not be expected because of the many barriers that the metabolites encounter when traveling from the blood stream to the enveloped cells and back to circulation. However, despite these diffusion barriers, many studies have shown optimal regulation, allowing us to conclude that encapsulated grafts do not always follow nature's course but are still a possible solution for many endocrine disorders for which the minute-to-minute regulation of metabolites is mandatory. Copyright © 2013 Elsevier B.V. All rights reserved.

Pancreatic β-Cell Dysfunction in Diet-Induced Obese Mice: Roles of AMP-Kinase, Protein Kinase Cε, Mitochondrial and Cholesterol Metabolism, and Alterations in Gene Expression

PubMed Central

Pepin, Émilie; Al-Mass, Anfal; Attané, Camille; Zhang, Kezhuo; Lamontagne, Julien; Lussier, Roxane; Madiraju, S. R. Murthy; Joly, Erik; Ruderman, Neil B.; Sladek, Robert; Prentki, Marc; Peyot, Marie-Line

2016-01-01

Diet induced obese (DIO) mice can be stratified according to their weight gain in response to high fat diet as low responders (LDR) and high responders (HDR). This allows the study of β-cell failure and the transitions to prediabetes (LDR) and early diabetes (HDR). C57BL/6N mice were fed for 8 weeks with a normal chow diet (ND) or a high fat diet and stratified as LDR and HDR. Freshly isolated islets from ND, LDR and HDR mice were studied ex-vivo for mitochondrial metabolism, AMPK activity and signalling, the expression and activity of key enzymes of energy metabolism, cholesterol synthesis, and mRNA profiling. Severely compromised glucose-induced insulin secretion in HDR islets, as compared to ND and LDR islets, was associated with suppressed AMP-kinase activity. HDR islets also showed reduced acetyl-CoA carboxylase activity and enhanced activity of 3-hydroxy-3-methylglutaryl-CoA reductase, which led respectively to elevated fatty acid oxidation and increased cholesterol biosynthesis. HDR islets also displayed mitochondrial membrane hyperpolarization and reduced ATP turnover in the presence of elevated glucose. Expression of protein kinase Cε, which reduces both lipolysis and production of signals for insulin secretion, was elevated in DIO islets. Genes whose expression increased or decreased by more than 1.2-fold were minor between LDR and ND islets (17 differentially expressed), but were prominent between HDR and ND islets (1508 differentially expressed). In HDR islets, particularly affected genes were related to cell cycle and proliferation, AMPK signaling, mitochondrial metabolism and cholesterol metabolism. In conclusion, chronically reduced AMPK activity, mitochondrial dysfunction, elevated cholesterol biosynthesis in islets, and substantial alterations in gene expression accompany β-cell failure in HDR islets. The β-cell compensation process in the prediabetic state (LDR) is largely independent of transcriptional adaptive changes, whereas the transition to early diabetes (HDR) is associated with major alterations in gene expression. PMID:27043434

Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

PubMed Central

Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

2014-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

PubMed Central

Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

2009-01-01

Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

Efficacy of DHMEQ, a NF-κB inhibitor, in islet transplantation: II. Induction DHMEQ treatment ameliorates subsequent alloimmune responses and permits long-term islet allograft acceptance.

PubMed

Watanabe, Masaaki; Yamashita, Kenichiro; Kamachi, Hirofumi; Kuraya, Daisuke; Koshizuka, Yasuyuki; Shibasaki, Susumu; Asahi, Yoh; Ono, Hitoshi; Emoto, Shin; Ogura, Masaomi; Yoshida, Tadashi; Ozaki, Michitaka; Umezawa, Kazuo; Matsushita, Michiaki; Todo, Satoru

2013-09-15

Long-term graft deterioration remains a major obstacle in the success of pancreatic islet transplantation (PITx). Antigen-independent inflammatory and innate immune responses strengthen subsequent antigen-dependent immunity; further, activation of nuclear factor (NF)-κB plays a key role during these responses. In this study, we tested our hypothesis that, by the inhibition of NF-κB activation, the suppression of these early responses after PITx could facilitate graft acceptance. Full major histocompatibility complex (MHC)-mismatched BALB/c (H-2) mice islets were transplanted into streptozotocin-induced diabetic C57BL/6 (B6: H-2) mice. The NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) was administered for either 3 or 14 days after PITx. To some PITx recipients, tacrolimus was also administered. Islet allograft survival, alloimmune responses, and in vitro effects of DHMEQ on dendritic cells (DCs) were assessed. With a vehicle treatment, 600 islet allografts were promptly rejected after PITx. In contrast, 3-day treatment with DHMEQ, followed by 2-week treatment with tacrolimus, allowed permanent acceptance of islet allografts. The endogenous danger-signaling molecule high mobility group complex 1 (HMGB1) was elevated in sera shortly after PITx, whereas DHMEQ administration abolished this elevation. DHMEQ suppressed HMGB1-driven cellular activation and proinflammatory cytokine secretion in mouse bone marrow-derived DCs and significantly reduced the capacity of DCs to prime allogeneic T-cell proliferation in vitro. Finally, the DHMEQ plus tacrolimus regimen reverted the diabetic state with only 300 islet allografts. Inhibition of NF-κB activation by DHMEQ shortly after PITx suppresses HMGB1, which activates DCs and strengthens the magnitude of alloimmune responses; this permits long-term islet allograft acceptance, even in case of fewer islet allografts.

Engraftment versus immunosuppression: cost-benefit analysis of immunosuppression after intrahepatic murine islet transplantation.

PubMed

Marzorati, Simona; Melzi, Raffaella; Citro, Antonio; Cantarelli, Elisa; Mercalli, Alessia; Scavini, Marina; Piemonti, Lorenzo

2014-05-27

Immunosuppression (IS) in islet transplantation (Tx) is a double-edged sword: it prevents immunoreaction but has the potential to impair islet engraftment. The aim of this study was to identify in murine animal models the IS platform with the best balance between these two opposite effects. To study the impact of IS on islet engraftment diabetic C57BL/6 mice were transplanted with 350 syngeneic islets through the portal vein and treated once-daily with either rapamycin (RAPA; 0.1-0.5-1 mg/kg ip), tacrolimus (FK506; 0.1-0.5-1 mg/kg ip), mycophenolate mofetil (MMF; 60-120-300 mg/kg oral) or vehicle for 14 days. Islet function was evaluated by measuring not-fasting glycemia and by performing an IVGTT on days 15 and 30 post-Tx. RAPA ≥0.5 mg/Kg, FK506 ≥0.5 mg/Kg, and MMF ≥120 mg/kg had detrimental effects on islet engraftment but not on the function of islets already engrafted in the liver. The effect on engraftment was irreversible and persisted even after IS withdrawal. The lower dose of IS that did not affect engraftment was tested for preventing rejection in the full mismatch allogeneic Tx BALB/c to C57BL/6 model. RAPA and/or FK506 were inefficient in preventing rejection, even when anti-IL2R mAb was added to the IS regimen. On the other hand, MMF alone or in association with FK506 significantly prolonged the time to islet rejection. IS showed profound dose-dependent deleterious effects on islet cell engraftment. The MMF/FK506 combination proved the best balance with less toxicity at the time of engraftment and more efficacy in controlling graft rejection.

Glycemic Stability Through Islet-After-Kidney Transplantation Using an Alemtuzumab-Based Induction Regimen and Long-Term Triple-Maintenance Immunosuppression.

PubMed

Nijhoff, M F; Engelse, M A; Dubbeld, J; Braat, A E; Ringers, J; Roelen, D L; van Erkel, A R; Spijker, H S; Bouwsma, H; van der Boog, P J M; de Fijter, J W; Rabelink, T J; de Koning, E J P

2016-01-01

Pancreatic islet transplantation is performed in a select group of patients with type 1 diabetes mellitus. Immunosuppressive regimens play an important role in long-term islet function. We aimed to investigate the efficacy of islet transplantation in patients with type 1 diabetes and a previous kidney transplantation using an alemtuzumab-based induction regimen and triple maintenance immunosuppression. Patients with type 1 diabetes, who had received a kidney transplant previously, were treated with alemtuzumab as induction therapy for their first islet transplantation and basiliximab induction therapy for subsequent islet transplantations. Maintenance immunosuppression consisted of triple immunosuppression (tacrolimus, mycophenolate mofetil, and prednisolone). Thirteen patients (age 50.9 ± 9.2 years, duration of diabetes 35 ± 9 years) received a total of 22 islet transplantations. One- and 2-year insulin independence was 62% and 42%, respectively; graft function was 100% and 92%, respectively. HbA1c dropped from 57.2 ± 13.1 (7.4 ± 1.2%) to 44.5 ± 11.8 mmol/molHb (6.2 ± 0.9%) (p = 0.003) after 2 years. Six of 13 patients suffered from severe hypoglycemia before islet transplantation. After transplantation, severe hypoglycemia was restricted to the only patient who lost graft function. Creatinine clearance was unchanged. Islet-after-kidney transplantation in patients with type 1 diabetes using an alemtuzumab-based induction regimen leads to considerable islet allograft function and improvement in glycemic control. © Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.

Use of additives, scaffolds and extracellular matrix components for improvement of human pancreatic islet outcomes in vitro: A systematic review.

PubMed

Lemos, Natália Emerim; de Almeida Brondani, Letícia; Dieter, Cristine; Rheinheimer, Jakeline; Bouças, Ana Paula; Bauermann Leitão, Cristiane; Crispim, Daisy; Bauer, Andrea Carla

2017-09-03

Pancreatic islet transplantation is an established treatment to restore insulin independence in type 1 diabetic patients. Its success rates have increased lately based on improvements in immunosuppressive therapies and on islet isolation and culture. It is known that the quality and quantity of viable transplanted islets are crucial for the achievement of insulin independence and some studies have shown that a significant number of islets are lost during culture time. Thus, in an effort to improve islet yield during culture period, researchers have tested a variety of additives in culture media as well as alternative culture devices, such as scaffolds. However, due to the use of different categories of additives or devices, it is difficult to draw a conclusion on the benefits of these strategies. Therefore, the aim of this systematic review was to summarize the results of studies that described the use of medium additives, scaffolds or extracellular matrix (ECM) components during human pancreatic islets culture. PubMed and Embase repositories were searched. Of 5083 articles retrieved, a total of 37 articles fulfilled the eligibility criteria and were included in the review. After data extraction, articles were grouped as follows: 1) "antiapoptotic/anti-inflammatory/antioxidant," 2) "hormone," 3) "sulphonylureas," 4) "serum supplements," and 5) "scaffolds or ECM components." The effects of the reviewed additives, ECM or scaffolds on islet viability, apoptosis and function (glucose-stimulated insulin secretion - GSIS) were heterogeneous, making any major conclusion hard to sustain. Overall, some "antiapoptotic/anti-inflammatory/antioxidant" additives decreased apoptosis and improved GSIS. Moreover, islet culture with ECM components or scaffolds increased GSIS. More studies are needed to define the real impact of these strategies in improving islet transplantation outcomes.

Improvement in Outcomes of Clinical Islet Transplantation: 1999–2010

PubMed Central

Barton, Franca B.; Rickels, Michael R.; Alejandro, Rodolfo; Hering, Bernhard J.; Wease, Stephen; Naziruddin, Bashoo; Oberholzer, Jose; Odorico, Jon S.; Garfinkel, Marc R.; Levy, Marlon; Pattou, Francois; Berney, Thierry; Secchi, Antonio; Messinger, Shari; Senior, Peter A.; Maffi, Paola; Posselt, Andrew; Stock, Peter G.; Kaufman, Dixon B.; Luo, Xunrong; Kandeel, Fouad; Cagliero, Enrico; Turgeon, Nicole A.; Witkowski, Piotr; Naji, Ali; O’Connell, Philip J.; Greenbaum, Carla; Kudva, Yogish C.; Brayman, Kenneth L.; Aull, Meredith J.; Larsen, Christian; Kay, Tom W.H.; Fernandez, Luis A.; Vantyghem, Marie-Christine; Bellin, Melena; Shapiro, A.M. James

2012-01-01

OBJECTIVE To describe trends of primary efficacy and safety outcomes of islet transplantation in type 1 diabetes recipients with severe hypoglycemia from the Collaborative Islet Transplant Registry (CITR) from 1999 to 2010. RESEARCH DESIGN AND METHODS A total of 677 islet transplant-alone or islet-after-kidney recipients with type 1 diabetes in the CITR were analyzed for five primary efficacy outcomes and overall safety to identify any differences by early (1999–2002), mid (2003–2006), or recent (2007–2010) transplant era based on annual follow-up to 5 years. RESULTS Insulin independence at 3 years after transplant improved from 27% in the early era (1999–2002, n = 214) to 37% in the mid (2003–2006, n = 255) and to 44% in the most recent era (2007–2010, n = 208; P = 0.006 for years-by-era; P = 0.01 for era alone). C-peptide ≥0.3 ng/mL, indicative of islet graft function, was retained longer in the most recent era (P < 0.001). Reduction of HbA1c and resolution of severe hypoglycemia exhibited enduring long-term effects. Fasting blood glucose stabilization also showed improvements in the most recent era. There were also modest reductions in the occurrence of adverse events. The islet reinfusion rate was lower: 48% by 1 year in 2007–2010 vs. 60–65% in 1999–2006 (P < 0.01). Recipients that ever achieved insulin-independence experienced longer duration of islet graft function (P < 0.001). CONCLUSIONS The CITR shows improvement in primary efficacy and safety outcomes of islet transplantation in recipients who received transplants in 2007–2010 compared with those in 1999–2006, with fewer islet infusions and adverse events per recipient. PMID:22723582

Effect of the Purinergic Inhibitor Oxidized ATP in a Model of Islet Allograft Rejection

PubMed Central

Vergani, Andrea; Fotino, Carmen; D’Addio, Francesca; Tezza, Sara; Podetta, Michele; Gatti, Francesca; Chin, Melissa; Bassi, Roberto; Molano, Ruth D.; Corradi, Domenico; Gatti, Rita; Ferrero, Maria E.; Secchi, Antonio; Grassi, Fabio; Ricordi, Camillo; Sayegh, Mohamed H.; Maffi, Paola; Pileggi, Antonello; Fiorina, Paolo

2013-01-01

The lymphocytic ionotropic purinergic P2X receptors (P2X1R-P2X7R, or P2XRs) sense ATP released during cell damage-activation, thus regulating T-cell activation. We aim to define the role of P2XRs during islet allograft rejection and to establish a novel anti-P2XRs strategy to achieve long-term islet allograft function. Our data demonstrate that P2X1R and P2X7R are induced in islet allograft-infiltrating cells, that only P2X7R is increasingly expressed during alloimmune response, and that P2X1R is augmented in both allogeneic and syngeneic transplantation. In vivo short-term P2X7R targeting (using periodate-oxidized ATP [oATP]) delays islet allograft rejection, reduces the frequency of Th1/Th17 cells, and induces hyporesponsiveness toward donor antigens. oATP-treated mice displayed preserved islet grafts with reduced Th1 transcripts. P2X7R targeting and rapamycin synergized in inducing long-term islet function in 80% of transplanted mice and resulted in reshaping of the recipient immune system. In vitro P2X7R targeting using oATP reduced T-cell activation and diminished Th1/Th17 cytokine production. Peripheral blood mononuclear cells obtained from long-term islet-transplanted patients showed an increased percentage of P2X7R+CD4+ T cells compared with controls. The beneficial effects of oATP treatment revealed a role for the purinergic system in islet allograft rejection, and the targeting of P2X7R is a novel strategy to induce long-term islet allograft function. PMID:23315496

Three-dimensional image study on the vascular structure after angiopoietin-1 transduction in isolated mouse pancreatic islets

NASA Astrophysics Data System (ADS)

He, Jing; Su, Dongming; Trucco, Massimo

2008-02-01

Angiopoietin-1 (Ang-1) is essential for remodeling the primitive vascular plexus during embryonic development and for reducing plasma leakage in inflammation of adult vasculature. However, the role for Ang-1 in maintenance of vascular stability in isolated pancreatic islets is not fully understood. In this study, we compared the difference of vascular morphology between Ang-1 treated (n=5) and control mouse islets (n=5) using both two- and three-dimensional optical image analysis. Isolated mouse islets were transduced with Ang-1 or Lac Z (control) vector at 37°C for 16 hours. Islets were incubated with both rat anti-CD31 antibody and rabbit anti-insulin antibody followed by incubation with Rhodamine-conjugated goat anti-rat IgG and Alexa-488 conjugated goat anti-rabbit IgG. Islets were viewed under a Nikon confocal microscope. Serial optical section images were captured and reconstructed using Nikon EZ-C1 software. Individual two-D and reconstructed three-D images were analyzed using MetaMorph Image Analysis software. Islet vascular density was determined. In two-D images, there was no significant difference of vascular density between the two groups. The vascular morphology didn't show any obvious differences in two-D images either. However, in the three-D images, we found higher vascular density and more vascular branches in the Ang-1 transducted islets and vascular dilation in control group. In conclusion, using three-D image analysis, Ang-1 displayed functions in maintenance of vascular stability and in stimulating growth of vascular branches in isolated mouse pancreatic islets. In order to study further the regeneration of different cell contents in the spherical pancreatic islet, three-D image analysis is an effective method to approach this goal.

Human islet cells are killed by BID-independent mechanisms in response to FAS ligand.

PubMed

Joglekar, Mugdha V; Trivedi, Prerak M; Kay, Thomas W; Hawthorne, Wayne J; O'Connell, Philip J; Jenkins, Alicia J; Hardikar, Anandwardhan A; Thomas, Helen E

2016-04-01

Cell death via FAS/CD95 can occur either by activation of caspases alone (extrinsic) or by activation of mitochondrial death signalling (intrinsic) depending on the cell type. The BH3-only protein BID is activated in the BCL-2-regulated or mitochondrial apoptosis pathway and acts as a switch between the extrinsic and intrinsic cell death pathways. We have previously demonstrated that islets from BID-deficient mice are protected from FAS ligand-mediated apoptosis in vitro. However, it is not yet known if BID plays a similar role in human beta cell death. We therefore aimed to test the role of BID in human islet cell apoptosis immediately after isolation from human cadaver donors, as well as after de-differentiation in vitro. Freshly isolated human islets or 10-12 day cultured human islet cells exhibited BID transcript knockdown after BID siRNA transfection, however they were not protected from FAS ligand-mediated cell death in vitro as determined by DNA fragmentation analysis using flow cytometry. On the other hand, the same cells transfected with siRNA for FAS-associated via death domain (FADD), a molecule in the extrinsic cell death pathway upstream of BID, showed significant reduction in cell death. De-differentiated islets (human islet-derived progenitor cells) also demonstrated similar results with no difference in cell death after BID knockdown as compared to scramble siRNA transfections. Our results indicate that BID-independent pathways are responsible for FAS-dependent human islet cell death. These results are different from those observed in mouse islets and therefore demonstrate potentially alternate pathways of FAS ligand-induced cell death in human and mouse islet cells.

Encapsulated Glucagon-Like Peptide-1-Producing Mesenchymal Stem Cells Have a Beneficial Effect on Failing Pig Hearts

PubMed Central

Wright, Elizabeth J.; Farrell, Kelly A.; Malik, Nadim; Kassem, Moustapha; Lewis, Andrew L.; Wallrapp, Christine

2012-01-01

Stem cell therapy is an exciting and emerging treatment option to promote post-myocardial infarction (post-MI) healing; however, cell retention and efficacy in the heart remain problematic. Glucagon-like peptide-1 (GLP-1) is an incretin hormone with cardioprotective properties but a short half-life in vivo. The effects of prolonged GLP-1 delivery from stromal cells post-MI were evaluated in a porcine model. Human mesenchymal stem cells immortalized and engineered to produce a GLP-1 fusion protein were encapsulated in alginate (bead-GLP-1 MSC) and delivered to coronary artery branches. Control groups were cell-free beads and beads containing unmodified MSCs (bead-MSC), n = 4–5 per group. Echocardiography confirmed left ventricular (LV) dysfunction at time of delivery in all groups. Four weeks after intervention, only the bead-GLP-1 MSC group demonstrated LV function improvement toward baseline and showed decreased infarction area compared with controls. Histological analysis showed reduced inflammation and a trend toward reduced apoptosis in the infarct zone. Increased collagen but fewer myofibroblasts were observed in infarcts of the bead-GLP-1 MSC and bead-MSC groups, and significantly more vessels per mm2 were noted in the infarct of the bead-GLP-1 MSC group. No differences were observed in myocyte cross-sectional area between groups. Post-MI delivery of GLP-1 encapsulated genetically modified MSCs provided a prolonged supply of GLP-1 and paracrine stem cell factors, which improved LV function and reduced epicardial infarct size. This was associated with increased angiogenesis and an altered remodeling response. Combined benefits of paracrine stem cell factors and GLP-1 were superior to those of stem cells alone. These results suggest that encapsulated genetically modified MSCs would be beneficial for recovery following MI. PMID:23197668

Using the cost-effectiveness of allogeneic islet transplantation to inform induced pluripotent stem cell-derived β-cell therapy reimbursement.

PubMed

Archibald, Peter R T; Williams, David J

2015-11-01

In the present study a cost-effectiveness analysis of allogeneic islet transplantation was performed and the financial feasibility of a human induced pluripotent stem cell-derived β-cell therapy was explored. Previously published cost and health benefit data for islet transplantation were utilized to perform the cost-effectiveness and sensitivity analyses. It was determined that, over a 9-year time horizon, islet transplantation would become cost saving and 'dominate' the comparator. Over a 20-year time horizon, islet transplantation would incur significant cost savings over the comparator (GB£59,000). Finally, assuming a similar cost of goods to islet transplantation and a lack of requirement for immunosuppression, a human induced pluripotent stem cell-derived β-cell therapy would dominate the comparator over an 8-year time horizon.

B7-H4 as a protective shield for pancreatic islet beta cells.

PubMed

Sun, Annika C; Ou, Dawei; Luciani, Dan S; Warnock, Garth L

2014-12-15

Auto- and alloreactive T cells are major culprits that damage β-cells in type 1 diabetes (T1D) and islet transplantation. Current immunosuppressive drugs can alleviate immune-mediated attacks on islets. T cell co-stimulation blockade has shown great promise in autoimmunity and transplantation as it solely targets activated T cells, and therefore avoids toxicity of current immunosuppressive drugs. An attractive approach is offered by the newly-identified negative T cell co-signaling molecule B7-H4 which is expressed in normal human islets, and its expression co-localizes with insulin. A concomitant decrease in B7-H4/insulin co-localization is observed in human type 1 diabetic islets. B7-H4 may play protective roles in the pancreatic islets, preserving their function and survival. In this review we outline the protective effect of B7-H4 in the contexts of T1D, islet cell transplantation, and potentially type 2 diabetes. Current evidence offers encouraging data regarding the role of B7-H4 in reversal of autoimmune diabetes and donor-specific islet allograft tolerance. Additionally, unique expression of B7-H4 may serve as a potential biomarker for the development of T1D. Future studies should continue to focus on the islet-specific effects of B7-H4 with emphasis on mechanistic pathways in order to promote B7-H4 as a potential therapy and cure for T1D.

Human pancreatic islet transplantation: an update and description of the establishment of a pancreatic islet isolation laboratory.

PubMed

Rheinheimer, Jakeline; Bauer, Andrea C; Silveiro, Sandra P; Estivalet, Aline A F; Bouças, Ana P; Rosa, Annelise R; Souza, Bianca M de; Oliveira, Fernanda S de; Cruz, Lavínia A; Brondani, Letícia A; Azevedo, Mirela J; Lemos, Natália E; Carlessi, Rodrigo; Assmann, Taís S; Gross, Jorge L; Leitão, Cristiane B; Crispim, Daisy

2015-04-01

Type 1 diabetes mellitus (T1DM) is associated with chronic complications that lead to high morbidity and mortality rates in young adults of productive age. Intensive insulin therapy has been able to reduce the likelihood of the development of chronic diabetes complications. However, this treatment is still associated with an increased incidence of hypoglycemia. In patients with "brittle T1DM", who have severe hypoglycemia without adrenergic symptoms (hypoglycemia unawareness), islet transplantation may be a therapeutic option to restore both insulin secretion and hypoglycemic perception. The Edmonton group demonstrated that most patients who received islet infusions from more than one donor and were treated with steroid-free immunosuppressive drugs displayed a considerable decline in the initial insulin independence rates at eight years following the transplantation, but showed permanent C-peptide secretion, which facilitated glycemic control and protected patients against hypoglycemic episodes. Recently, data published by the Collaborative Islet Transplant Registry (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully decrease plasma glucose and HbA1c levels, the occurrence of severe hypoglycemia, and improve patient quality of life. The goal of this paper was to review the human islet isolation and transplantation processes, and to describe the establishment of a human islet isolation laboratory at the Endocrine Division of the Hospital de Clínicas de Porto Alegre - Rio Grande do Sul, Brazil.

Bioengineering the Endocrine Pancreas: Intraomental Islet Transplantation Within a Biologic Resorbable Scaffold.

PubMed

Berman, Dora M; Molano, R Damaris; Fotino, Carmen; Ulissi, Ulisse; Gimeno, Jennifer; Mendez, Armando J; Kenyon, Norman M; Kenyon, Norma S; Andrews, David M; Ricordi, Camillo; Pileggi, Antonello

2016-05-01

Transplantation of pancreatic islets is a therapeutic option to preserve or restore β-cell function. Our study was aimed at developing a clinically applicable protocol for extrahepatic transplantation of pancreatic islets. The potency of islets implanted onto the omentum, using an in situ-generated adherent, resorbable plasma-thrombin biologic scaffold, was evaluated in diabetic rat and nonhuman primate (NHP) models. Intraomental islet engraftment in the biologic scaffold was confirmed by achievement of improved metabolic function and preservation of islet cytoarchitecture, with reconstitution of rich intrainsular vascular networks in both species. Long-term nonfasting normoglycemia and adequate glucose clearance (tolerance tests) were achieved in both intrahepatic and intraomental sites in rats. Intraomental graft recipients displayed lower levels of serum biomarkers of islet distress (e.g., acute serum insulin) and inflammation (e.g., leptin and α2-macroglobulin). Importantly, low-purity (30:70% endocrine:exocrine) syngeneic rat islet preparations displayed function equivalent to that of pure (>95% endocrine) preparations after intraomental biologic scaffold implantation. Moreover, the biologic scaffold sustained allogeneic islet engraftment in immunosuppressed recipients. Collectively, our feasibility/efficacy data, along with the simplicity of the procedure and the safety of the biologic scaffold components, represented sufficient preclinical testing to proceed to a pilot phase I/II clinical trial. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Phase transitions in pancreatic islet cellular networks and implications for type-1 diabetes

NASA Astrophysics Data System (ADS)

Stamper, I. J.; Jackson, Elais; Wang, Xujing

2014-01-01

In many aspects the onset of a chronic disease resembles a phase transition in a complex dynamic system: Quantitative changes accumulate largely unnoticed until a critical threshold is reached, which causes abrupt qualitative changes of the system. In this study we examine a special case, the onset of type-1 diabetes (T1D), a disease that results from loss of the insulin-producing pancreatic islet β cells. Within each islet, the β cells are electrically coupled to each other via gap-junctional channels. This intercellular coupling enables the β cells to synchronize their insulin release, thereby generating the multiscale temporal rhythms in blood insulin that are critical to maintaining blood glucose homeostasis. Using percolation theory we show how normal islet function is intrinsically linked to network connectivity. In particular, the critical amount of β-cell death at which the islet cellular network loses site percolation is consistent with laboratory and clinical observations of the threshold loss of β cells that causes islet functional failure. In addition, numerical simulations confirm that the islet cellular network needs to be percolated for β cells to synchronize. Furthermore, the interplay between site percolation and bond strength predicts the existence of a transient phase of islet functional recovery after onset of T1D and introduction of treatment, potentially explaining the honeymoon phenomenon. Based on these results, we hypothesize that the onset of T1D may be the result of a phase transition of the islet β-cell network.

Impact of an autologous oxygenating matrix culture system on rat islet transplantation outcome.

PubMed

Schaschkow, A; Mura, C; Bietiger, W; Peronet, C; Langlois, A; Bodin, F; Dissaux, C; Bruant-Rodier, C; Pinget, M; Jeandidier, N; Juszczak, M T; Sigrist, S; Maillard, E

2015-06-01

Disruption of the pancreatic islet environment combined with the decrease in oxygen supply that occurs during isolation leads to poor islet survival. The aim of this study was to validate the benefit of using a plasma-based scaffold supplemented with perfluorodecalin to improve islet transplantation outcome. Rat islets were cultured in three conditions: i) control group, ii) plasma based-matrix (P-matrix), and iii) P-matrix supplemented with emulsified perfluorodecalin. After 24 h culture, matrix/cell contacts (Integrinβ1, p-FAK/FAK, p-Akt/Akt), survival (caspase 3, TUNEL, FDA/PI), function, and HIF-1α translocation were assessed. Afterwards, P-matrices were dissolved and the islets were intraportally transplanted. Graft function was monitored for 31 days with glycaemia and C-peptide follow up. Inflammation was assessed by histology (macrophage and granulocyte staining) and thrombin/anti-thrombin complex measurement. Islet survival correlated with an increase in integrin, FAK, and Akt activation in P-matrices and function was maintained. Perfluorodecalin supplementation decreased translocation of HIF-1α in the nucleus and post-transplantation islet structure was better preserved in P-matrices, but a quicker activation of IBMIR resulted in early loss of graft function. "Oxygenating" P-matrices provided a real benefit to islet survival and resistance in vivo. However, intraportal transplantation is not suitable for this kind of culture due to IBMIR; thus, alternative sites must be explored. Copyright © 2015 Elsevier Ltd. All rights reserved.

Topologically heterogeneous beta cell adaptation in response to high-fat diet in mice.

PubMed

Ellenbroek, Johanne H; Töns, Hendrica A; de Graaf, Natascha; Loomans, Cindy J; Engelse, Marten A; Vrolijk, Hans; Voshol, Peter J; Rabelink, Ton J; Carlotti, Françoise; de Koning, Eelco J

2013-01-01

Beta cells adapt to an increased insulin demand by enhancing insulin secretion via increased beta cell function and/or increased beta cell number. While morphological and functional heterogeneity between individual islets exists, it is unknown whether regional differences in beta cell adaptation occur. Therefore we investigated beta cell adaptation throughout the pancreas in a model of high-fat diet (HFD)-induced insulin resistance in mice. C57BL/6J mice were fed a HFD to induce insulin resistance, or control diet for 6 weeks. The pancreas was divided in a duodenal (DR), gastric (GR) and splenic (SR) region and taken for either histology or islet isolation. The capacity of untreated islets from the three regions to adapt in an extrapancreatic location was assessed by transplantation under the kidney capsule of streptozotocin-treated mice. SR islets showed 70% increased beta cell proliferation after HFD, whereas no significant increase was found in DR and GR islets. Furthermore, isolated SR islets showed twofold enhanced glucose-induced insulin secretion after HFD, as compared with DR and GR islets. In contrast, transplantation of islets isolated from the three regions to an extrapancreatic location in diabetic mice led to a similar decrease in hyperglycemia and no difference in beta cell proliferation. HFD-induced insulin resistance leads to topologically heterogeneous beta cell adaptation and is most prominent in the splenic region of the pancreas. This topological heterogeneity in beta cell adaptation appears to result from extrinsic factors present in the islet microenvironment.

Identification and molecular characterization of serological group C streptococci isolated from diseased pigs and monkeys in Indonesia.

PubMed Central

Soedarmanto, I; Pasaribu, F H; Wibawan, I W; Lämmler, C

1996-01-01

The present study was designed to comparatively investigate 34 beta-hemolytic streptococci isolated from infected pigs and monkeys from various islands in Indonesia. According to the serological and biochemical data, all 34 isolates were Lancefield's serological group C streptococci and could be identified as Streptococcus equi subsp. zooepidemicus. Of the 34 group C streptococci investigated, 28 grew on solid media in large, mucoid colonies, in fluid media at a uniform turbidity, and in soft agar in diffuse colonies. A decapsulation test with a hyaluronidase-producing Staphylococcus aureus strain revealed the hyaluronic acid nature of the capsular material. The remaining six streptococci grew on solid media in small, nonmucoid colonies, in fluid media as sediment with clear supernatant, and in soft agar in compact colonies. Determination of surface hydrophobicity by salt aggregation revealed a hydrophilic surface for the encapsulated bacteria and a hydrophobic surface for the unencapsulated group C streptococci. To further analyze the epidemiological relationships, all 34 mucoid and nonmucoid isolates from pigs and monkeys were subjected to protein and DNA fingerprinting. The latter was performed by pulsed-field gel electrophoresis. The protein profiles of all 34 isolates and the DNA profiles of 32 isolates appeared to be identical, with the DNA profiles of 2 isolates being closely related, indicating that a single virulent clone is responsible for this disease outbreak in Indonesia. PMID:8862585

Progress and Challenges in Macroencapsulation Approaches for Type 1 Diabetes (T1D) Treatment: Cells, Biomaterials, and Devices

PubMed Central

Song, Shang; Roy, Shuvo

2018-01-01

Macroencapsulation technology has been an attractive topic in the field of treatment for Type 1 diabetes due to mechanical stability, versatility, and retrievability of the macrocapsule design. Macro-capsules can be categorized into extravascular and intravascular devices, in which solute transport relies either on diffusion or convection, respectively. Failure of macroencapsulation strategies can be due to limited regenerative capacity of the encased insulin-producing cells, sub-optimal performance of encapsulation biomaterials, insufficient immunoisolation, excessive blood thrombosis for vascular perfusion devices, and inadequate modes of mass transfer to support cell viability and function. However, significant technical advancements have been achieved in macroencapsulation technology, namely reducing diffusion distance for oxygen and nutrients, using pro-angiogenic factors to increase vascularization for islet engraftment, and optimizing membrane permeability and selectivity to prevent immune attacks from host’s body. This review presents an overview of existing macroencapsulation devices and discusses the advances based on tissue-engineering approaches that will stimulate future research and development of macroencapsulation technology. PMID:26615050

Extracellular Matrix Scaffold Technology for Bioartificial Pancreas Engineering: State of the Art and Future Challenges.

PubMed

Salvatori, Marcus; Katari, Ravi; Patel, Timil; Peloso, Andrea; Mugweru, Jon; Owusu, Kofi; Orlando, Giuseppe

2014-01-01

Emergent technologies in regenerative medicine may soon overcome the limitations of conventional diabetes therapies. Collaborative efforts across the subfields of stem cell technology, islet encapsulation, and biomaterial carriers seek to produce a bioengineered pancreas capable of restoring endocrine function in patients with insulin-dependent diabetes. These technologies rely on a robust understanding of the extracellular matrix (ECM), the supportive 3-dimensional network of proteins necessary for cellular attachment, proliferation, and differentiation. Although these functions can be partially approximated by biosynthetic carriers, novel decellularization protocols have allowed researchers to discover the advantages afforded by the native pancreatic ECM. The native ECM has proven to be an optimal platform for recellularization and whole-organ pancreas bioengineering, an exciting new field with the potential to resolve the dire shortage of transplantable organs. This review seeks to contextualize recent findings, discuss current research goals, and identify future challenges of regenerative medicine as it applies to diabetes management. © 2014 Diabetes Technology Society.

Effects of Energy Dissipation Rate on Islets of Langerhans: Implications for Isolation and Transplantation

PubMed Central

Shenkman, Rustin M.; Godoy-Silva, Ruben; Papas, Klearchos K.; Chalmers, Jeffrey J.

2010-01-01

Acute physical stresses can occur in the procurement and isolation process and potentially can contribute to islet death or malfunction upon transplantation. A contractional flow device, previously used to subject suspended cells to well-defined hydrodynamic forces, has been modified and used to assess the vulnerability of porcine islets of Langerhans to hydrodynamic forces. The flow profiles and velocity gradients in this modified device were modeled using commercial CFD software and characterized, as in previous studies, with the scalar parameter, energy dissipation rate (EDR). Porcine islets were stressed in a single pass at various stress levels (i.e., values of EDR). Membrane integrity, oxygen uptake rate, caspase 3/7 activity, and insulin release were not affected by the levels of fluid stress tested up to an EDR of 2 × 103 W/m3. Visual observation of the stressed islets suggested that cells at the islet exterior were peeled away at EDR greater than 10,000 W/m3, however, this observation could not be confirmed using image analysis software, which determined the ratio of surface perimeter to total area. The result of this study suggests an upper limit in fluid stress to which islets can be subjected. Such upper limits assist in the design and operation of future islet processing equipment and processes. PMID:19191351

Correlation of pancreatic histopathologic findings and islet yield in children with chronic pancreatitis undergoing total pancreatectomy and islet autotransplantation.

PubMed

Kobayashi, Takashi; Manivel, Juan C; Bellin, Melena D; Carlson, Annelisa M; Moran, Antoinette; Freeman, Martin L; Hering, Bernhard J; Sutherland, David E R

2010-01-01

The probability of insulin independence after intraportal islet autotransplantation (IAT) for chronic pancreatitis (CP) treated by total pancreatectomy (TP) relates to the number of islets isolated from the excised pancreas. Our goal was to correlate the islet yield with the histopathologic findings and the clinical parameters in pediatric (age, <19 years) CP patients undergoing TP-IAT. Eighteen pediatric CP patients aged 5 to 18 years (median, 15.6 years) who underwent TP-IAT were studied. Demographics and clinical history came from medical records. Histopathologic specimens from the pancreas were evaluated for presence and severity of fibrosis, acinar cell atrophy, inflammation, and nesidioblastosis by a surgical pathologist blinded to clinical information. Fibrosis and acinar atrophy negatively correlated with islet yield (P = 0.02, r = -0.50), particularly in hereditary CP (P = 0.01). Previous duct drainage surgeries also had a strong negative correlation (P = 0.01). Islet yield was better in younger (preteen) children (P = 0.02, r = -0.61) and in those with pancreatitis of shorter duration (P = 0.04, r = -0.39). For preserving beta cell mass, it is best to perform TP-IAT early in the course of CP in children, and prior drainage procedures should be avoided to maximize the number of islets available, especially in hereditary disease.

Glycolytic and mitochondrial metabolism in pancreatic islets from MSG-treated obese rats subjected to swimming training.

PubMed

Leite, Nayara de Carvalho; Ferreira, Thiago Rentz; Rickli, Sarah; Borck, Patricia Cristine; Mathias, Paulo Cezar de Freitas; Emilio, Henriette Rosa de Oliveira; Grassiolli, Sabrina

2013-01-01

Obese rats obtained by neonatal monosodium glutamate (MSG) administration present insulin hypersecretion. The metabolic mechanism by which glucose catabolism is coupled to insulin secretion in the pancreatic β-cells from MSG-treated rats is understood. The purpose of this study was to evaluate glucose metabolism in pancreatic islets from MSG-treated rats subjected to swimming training. MSG-treated and control (CON) rats swam for 30 minutes (3 times/week) over a period of 10 weeks. Pancreatic islets were isolated and incubated with glucose in the presence of glycolytic or mitochondrial inhibitors. Swimming training attenuated fat pad accumulation, avoiding changes in the plasma levels of lipids, glucose and insulin in MSG-treated rats. Adipocyte and islet hypertrophy observed in MSG-treated rats were attenuated by exercise. Pancreatic islets from MSG-treated obese rats also showed insulin hypersecretion, greater glucose transporter 2 (GLUT2) expression, increased glycolytic flux and reduced mitochondrial complex III activity. Swimming training attenuated islet hypertrophy and normalised GLUT2 expression, contributing to a reduction in the glucose responsiveness of pancreatic islets from MSG-treated rats without altering glycolytic flux. However, physical training increased the activity of mitochondrial complex III in pancreatic islets from MSG-treated rats without a subsequent increase in glucose-induced insulin secretion. Copyright © 2013 S. Karger AG, Basel.

Quantitative Raman spectral changes of the differentiation of mesenchymal stem cells into islet-like cells by biochemical component analysis and multiple peak fitting

NASA Astrophysics Data System (ADS)

Su, Xin; Fang, Shaoyin; Zhang, Daosen; Zhang, Qinnan; He, Yingtian; Lu, Xiaoxu; Liu, Shengde; Zhong, Liyun

2015-12-01

Mesenchymal stem cells (MSCs) differentiate into islet-like cells, providing a possible solution for type I diabetes treatment. To search for the precise molecular mechanism of the directional differentiation of MSC-derived islet-like cells, biomolecular composition, and structural conformation information during MSC differentiation, is required. Because islet-like cells lack specific surface markers, the commonly employed immunostaining technique is not suitable for their identification, physical separation, and enrichment. Combining Raman spectroscopic data, a fitting accuracy-improved biochemical component analysis, and multiple peaks fitting approach, we identified the quantitative biochemical and intensity change of Raman peaks that show the differentiation of MSCs into islet-like cells. Along with increases in protein and glycogen content, and decreases in deoxyribonucleic acid and ribonucleic acid content, in islet-like cells relative to MSCs, it was found that a characteristic peak of insulin (665 cm-1) has twice the intensity in islet-like cells relative to MSCs, indicating differentiation of MSCs into islet-like cells was successful. Importantly, these Raman signatures provide useful information on the structural and pathological states during MSC differentiation and help to develop noninvasive and label-free Raman sorting methods for stem cells and their lineages.

Photoacoustic imaging of angiogenesis in subdermal islet transplant sites

NASA Astrophysics Data System (ADS)

Shi, Wei; Pawlick, Rena; Bruni, Antonio; Rafiei, Yasmin; Pepper, Andrew R.; Gala-Lopez, Boris; Choi, Min; Malcolm, Andrew; Zemp, Roger J.; Shapiro, A. M. James

2016-03-01

Exogenous insulin administration is the mainstay treatment therapy for patients with Type-1 diabetes mellitus (T1DM). However, for select patients, clinical islet transplantation is an alternative therapeutic treatment. In this procedure, islets are transplanted into the hepatic portal vein, and despite improved success within the last decade, obstacles are still associated with this approach. It has been discovered that the subcutaneous space may be an effective alternative site for islet transplantation, and may provide advantages of easy access and potential for simple monitoring. The ability to monitor islet viability and the transplant microenvironment may be key to future success in islet transplantation. A subcutaneous device-less technique has been developed to facilitate angiogenesis in the islet transplant site, however, a method for monitoring the potential engraftment site have yet to be explored fully. Here we demonstrate the ability to track angiogenesis in mice with 1, 2, 3 and 4 weeks post-catheter implant on both sides of the abdomen using a FujiFilm VisualSonics Vevo-LAZR system. Quantitative analysis on vessel densities exhibited gradual vessel growth successfully induced by catheter implantation. Our study demonstrates the ability of employing photoacoustic and micro-ultrasound imaging to track angiogenesis around the catheter site prior to islet transplantation.

RNA Interference for improving the Outcome of Islet Transplantation

PubMed Central

Li, Feng; Mahato, Ram I

2010-01-01

Islet transplantation has the potential to cure type 1 diabetes. Despite recent therapeutic success, it is still not common because a large number of transpanted islets get damaged by multiple challenges including instant blood mediated inflammatory reaction, hypoxia/reperfusion injury, inflammatory cytokines, and immune rejection. RNA interference (RNAi) is an novel strategy to selectively degrade target mRNA. The use of RNAi technologies to downregulate the expression of harmful genes has the potential to improve the outcome of islet transplantation. The aim of this review is to gain a thorough understanding of biological obstacles to islet transplantation and discuss how to overcome these barriers using different RNAi technologies. This eventually will help improve islet survival and function post transplantaion. Chemically synthesized small interferring RNA (siRNA), vector based short haripin RNA (shRNA), and their critical design elements (such as sequences, promoters, backbone) are discussed. The application of combinatorial RNAi in islet transplantation is also discussed. Last but not the least, several delivery strategies for enhanced gene silencing are discussed, including chemical modification of siRNA, complex formation, bioconjugation, and viral vectors. PMID:21156190

Zinc as a paracrine effector in pancreatic islet cell death.

PubMed

Kim, B J; Kim, Y H; Kim, S; Kim, J W; Koh, J Y; Oh, S H; Lee, M K; Kim, K W; Lee, M S

2000-03-01

Because of a huge amount of Zn2+ in secretory granules of pancreatic islet beta-cells, Zn2+ released in certain conditions might affect the function or survival of islet cells. We studied potential paracrine effects of endogenous Zn2+ on beta-cell death. Zn2+ induced insulinoma/islet cell death in a dose-dependent manner. Chelation of released endogenous Zn2+ by CaEDTA significantly decreased streptozotocin (STZ)-induced islet cell death in an in vitro culture system simulating in vivo circumstances but not in the conventional culture system. Zn2+ chelation in vivo by continuous CaEDTA infusion significantly decreased the incidence of diabetes after STZ administration. N-(6-methoxy-quinolyl)-para-toluene-sulfonamide staining revealed that Zn2+ was densely deposited in degenerating islet cells 24 h after STZ treatment, which was decreased by CaEDTA infusion. We show here that Zn2+ is not a passive element for insulin storage but an active participant in islet cell death in certain conditions, which in time might contribute to the development of diabetes in aged people.

Intraportal islet transplantation: the impact of the liver microenvironment.

PubMed

Delaune, Vaihere; Berney, Thierry; Lacotte, Stéphanie; Toso, Christian

2017-03-01

The portal vein remains the preferred site for pancreatic islet transplantation due to its easy access and low morbidity. However, despite great progress in isolation and transplantation protocols over the past few years, it is still associated with the early loss of some 50-70% of transplanted islets. The complex liver microenvironment itself presumably plays an important role in this loss. The present review focuses on the specifics of the liver microenvironment, notably the localized hepatic ischemia/reperfusion injury following transplantation, the low oxygenation of the portal vein, the instant blood-mediated inflammatory reaction, the endogenous liver immune system, and the gut-liver axis, and how they can each have an impact on the transplanted islets. It identifies the potential, or already applied, clinical interventions for improving intraportal islet survival, and pinpoints those promising areas still lacking preclinical research. Future interventions on clinical intraportal islet transplantation need to take into account the global context of the liver microenvironment, with multi-point interventions being most likely to improve early islet survival and engraftment. © 2017 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.

PEGylated bilirubin nanoparticle as an anti-oxidative and anti-inflammatory demulcent in pancreatic islet xenotransplantation.

PubMed

Kim, Min Jun; Lee, Yonghyun; Jon, Sangyong; Lee, Dong Yun

2017-07-01

Transplanted islets suffer hypoxic stress, which leads to nonspecific inflammation. This is the major cause of islet graft failure during the early stage of intrahepatic islet transplantation. Although bilirubin has shown potent anti-oxidative and anti-inflammatory functions, its clinical applications have been limited due to its insolubility and short half-life. To overcome this problem, novel amphiphilic bilirubin nanoparticles are designed. Hydrophilic poly(ethylene glycol) (PEG) is conjugated to the hydrophobic bilirubin molecule. Then, the PEG-bilirubin conjugates form nanoparticles via self-assembly, i.e., so-called to BRNPs. BRNPs can protect islet cells not only from chemically induced oxidative stress by scavenging reactive oxygen species molecules, but also from activated macrophages by suppressing cytokine release. Importantly, in vivo experiments demonstrate that BRNP treatment can dramatically and significantly prolong islet graft survival compared to bilirubin treatment. In addition, immunohistochemical analysis shows BRNPs have potent anti-oxidative and anti-inflammatory capabilities. Collectively, novel BRNPs can be a new potent remedy for successful islet transplantation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Wave forcing and morphological changes of New Caledonia lagoon islets: Insights on their possible relations

NASA Astrophysics Data System (ADS)

Aucan, Jérôme; Vendé-Leclerc, Myriam; Dumas, Pascal; Bricquir, Marianne

2017-10-01

In the present study, we examine how waves may contribute to the morphological changes of islets in the New Caledonia lagoon. We collected in situ wave data to investigate their characteristics. Three types of waves are identified and quantified: (1) high-frequency waves generated within the lagoon, (2) low-frequency waves originating from swells in the Tasman Sea, and (3) infragravity waves. We found out that high-frequency waves are the dominant forcing on the islets during typical wind events throughout the year, while infragravity waves, likely generated by the breaking of low-frequency waves, dominate during seasonal swell events. During swell events, low-frequency waves can also directly propagate to the islets through channels across the barrier reef, or be tidally modulated across the barrier reef before reaching the islets. Topographic surveys and beach profiles on one islet indicate areas with seasonal morphological changes and other areas with longer, interannual or decadal, erosion patterns. Although more data are needed to validate this hypothesis, we suspect that a relation exists between wave forcing and morphological changes of the islets.

The hypothalamic satiety peptide CART is expressed in anorectic and non-anorectic pancreatic islet tumors and in the normal islet of Langerhans.

PubMed

Jensen, P B; Kristensen, P; Clausen, J T; Judge, M E; Hastrup, S; Thim, L; Wulff, B S; Foged, C; Jensen, J; Holst, J J; Madsen, O D

1999-03-26

The hypothalamic satiety peptide CART (cocaine and amphetamine regulated transcript) is expressed at high levels in anorectic rat glucagonomas but not in hypoglycemic insulinomas. However, a non-anorectic metastasis derived from the glucagonoma retained high CART expression levels and produced circulating CART levels comparable to that of the anorectic tumors. Moreover, distinct glucagonoma lines derived by stable HES-1 transfection of the insulinoma caused severe anorexia but retained low circulating levels of CART comparable to that of insulinoma bearing or control rats. Islet tumor associated anorexia and circulating CART levels are thus not correlated, and in line with this peripheral administration of CART (5-50 mg/kg) produced no effect on feeding behavior. In the rat two alternatively spliced forms of CART mRNA exist and quantitative PCR revealed expression of both forms in the hypothalamus, in the different islet tumors, and in the islets of Langerhans. Immunocytochemistry as well as in situ hybridization localized CART expression to the somatostatin producing islet D cell. A potential endocrine/paracrine role of islet CART remains to be clarified.

Islet transplantation versus insulin therapy in patients with type 1 diabetes with severe hypoglycaemia or poorly controlled glycaemia after kidney transplantation (TRIMECO): a multicentre, randomised controlled trial.

PubMed

Lablanche, Sandrine; Vantyghem, Marie-Christine; Kessler, Laurence; Wojtusciszyn, Anne; Borot, Sophie; Thivolet, Charles; Girerd, Sophie; Bosco, Domenico; Bosson, Jean-Luc; Colin, Cyrille; Tetaz, Rachel; Logerot, Sophie; Kerr-Conte, Julie; Renard, Eric; Penfornis, Alfred; Morelon, Emmanuel; Buron, Fanny; Skaare, Kristina; Grguric, Gwen; Camillo-Brault, Coralie; Egelhofer, Harald; Benomar, Kanza; Badet, Lionel; Berney, Thierry; Pattou, François; Benhamou, Pierre-Yves

2018-05-15

Islet transplantation is indicated for patients with type 1 diabetes with severe hypoglycaemia or after kidney transplantation. We did a randomised trial to assess the efficacy and safety of islet transplantation compared with insulin therapy in these patients. In this multicentre, open-label, randomised controlled trial, we randomly assigned (1:1) patients with type 1 diabetes at 15 university hospitals to receive immediate islet transplantation or intensive insulin therapy (followed by delayed islet transplantation). Eligible patients were aged 18-65 years and had severe hypoglycaemia or hypoglycaemia unawareness, or kidney grafts with poor glycaemic control. We used computer-generated randomisation, stratified by centre and type of patient. Islet recipients were scheduled to receive 11 000 islet equivalents per kg bodyweight in one to three infusions. The primary outcome was proportion of patients with a modified β-score (in which an overall score of 0 was not allocated when stimulated C-peptide was negative) of 6 or higher at 6 months after first islet infusion in the immediate transplantation group or 6 months after randomisation in the insulin group. The primary analysis included all patients who received the allocated intervention; safety was assessed in all patients who received islet infusions. This trial is registered with ClinicalTrials.gov, number NCT01148680, and is completed. Between July 8, 2010, and July 29, 2013, 50 patients were randomly assigned to immediate islet transplantation (n=26) or insulin treatment (n=24), of whom three (one in the immediate islet transplantation group and two in the insulin therapy group) did not receive the allocated intervention. Median follow-up was 184 days (IQR 181-186) in the immediate transplantation group and 185 days (172-201) in the insulin therapy group. At 6 months, 16 (64% [95% CI 43-82]) of 25 patients in the immediate islet transplantation group had a modified β-score of 6 or higher versus none (0% [0-15]) of the 22 patients in the insulin group (p<0·0001). At 12 months after first infusion, bleeding complications had occurred in four (7% [2-18]) of 55 infusions, and a decrease in median glomerular filtration rate from 90·5 mL/min (IQR 76·6-94·0) to 71·8 mL/min (59·0-89·0) was observed in islet recipients who had not previously received a kidney graft and from 63·0 mL/min (55·0-71·0) to 57·0 mL/min (45·5-65·1) in islet recipients who had previously received a kidney graft. For the indications assessed in this study, islet transplantation effectively improves metabolic outcomes. Although studies with longer-term follow-up are needed, islet transplantation seems to be a valid option for patients with severe, unstable type 1 diabetes who are not responding to intensive medical treatments. However, immunosuppression can affect kidney function, necessitating careful selection of patients. Programme Hospitalier de Recherche Clinique grant from the French Government. Copyright © 2018 Elsevier Ltd. All rights reserved.

General Information about Pancreatic Neuroendocrine Tumors (Islet Cell Tumors)

MedlinePlus

... Islet Cell Tumors) Treatment (PDQ®)–Patient Version General Information About Pancreatic Neuroendocrine Tumors (Islet Cell Tumors) Go ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

Clinical pancreatic islet transplantation.

PubMed

Shapiro, A M James; Pokrywczynska, Marta; Ricordi, Camillo

2017-05-01

Clinical pancreatic islet transplantation can be considered one of the safest and least invasive transplant procedures. Remarkable progress has occurred in both the technical aspects of islet cell processing and the outcomes of clinical islet transplantation. With >1,500 patients treated since 2000, this therapeutic strategy has moved from a curiosity to a realistic treatment option for selected patients with type 1 diabetes mellitus (that is, those with hypoglycaemia unawareness, severe hypoglycaemic episodes and glycaemic lability). This Review outlines the techniques required for human islet isolation, in vitro culture before the transplant and clinical islet transplantation, and discusses indications, optimization of recipient immunosuppression and management of adjunctive immunomodulatory and anti-inflammatory strategies. The potential risks, long-term outcomes and advances in treatment after the transplant are also discussed to further move this treatment towards becoming a more widely available option for patients with type 1 diabetes mellitus and eventually a potential cure.

Pancreatic Islet Responses to Metabolic Trauma

PubMed Central

Burke, Susan J.; Karlstad, Michael D.; Collier, J. Jason

2016-01-01

Carbohydrate, lipid, and protein metabolism are largely controlled by the interplay of various hormones, which includes those secreted by the pancreatic islets of Langerhans. While typically representing only 1–2% of the total pancreatic mass, the islets have a remarkable ability to adapt to disparate situations demanding a change in hormone release, such as peripheral insulin resistance. There are many different routes to the onset of insulin resistance, including obesity, lipodystrophy, glucocorticoid excess, and the chronic usage of atypical anti-psychotic drugs. All of these situations are coupled to an increase in pancreatic islet size, often with a corresponding increase in insulin production. These adaptive responses within the islets are ultimately intended to maintain glycemic control and to promote macronutrient homeostasis during times of stress. Herein, we review the consequences of specific metabolic trauma that lead to insulin resistance and the corresponding adaptive alterations within the pancreatic islets. PMID:26974425

Nitric oxide interferes with islet cell zinc homeostasis.

PubMed

Tartler, U; Kröncke, K D; Meyer, K L; Suschek, C V; Kolb-Bachofen, V

2000-12-01

Zinc is crucial for the biosynthesis, storage, and secretion of insulin in pancreatic islet cells. We have previously presented evidence that NO interferes with cellular Zn(2+) homeostasis and we therefore investigated the influence of chronic NO exposure on the labile islet cell Zn(2+) content. A strong fluorescence activity in a large islet cell subpopulation was found after staining with the Zn(2+)-specific fluorophore Zinquin. Culture for 24 h in the presence of nontoxic concentrations of the slow-releasing NO donor DETA/NO resulted in a significantly reduced Zn(2+)-dependent fluorescence. This appears to be islet specific as in endothelial cells DETA/NO exposure enhanced the Zn(2+)-dependent fluorescence activity in a concentration-dependent manner. These results suggest that NO interferes with cellular Zn(2+) homeostasis, which in islet cells is crucial for proper hormone delivery and thus special cell function. Copyright 2000 Academic Press.

Distribution of IL-1β immunoreactive cells in pancreatic biopsies from living volunteers with new-onset type 1 diabetes: comparison with donors without diabetes and with longer duration of disease.

PubMed

Reddy, Shiva; Krogvold, Lars; Martin, Charlton; Holland, Rebecca; Choi, Jaimin; Woo, Hannah; Wu, Fiona; Dahl-Jørgensen, Knut

2018-06-01

Although IL-1β is considered a key mediator of beta cell destruction, its cellular expression in islets during early type 1 diabetes remains unclear. We compared its expression in rare pancreatic biopsies from new-onset living volunteers with its expression in cadaveric pancreas sections from non-diabetic autoantibody-positive and -negative individuals and those with long-standing disease. Pancreatic biopsy sections from six new-onset living volunteers (group 1) and cadaveric sections from 13 non-diabetic autoantibody-negative donors (group 2), four non-diabetic autoantibody-positive donors (group 3) and nine donors with diabetes of longer duration (0.25-12 years of disease; group 4) were triple-immunostained for IL-1β, insulin and glucagon. Intra- and peri-islet IL-1β-positive cells in insulin-positive and -negative islets and in random exocrine fields were enumerated. The mean number of IL-1β-positive cells per islet from each donor in peri- and intra-islet regions was <1.25 and <0.5, respectively. In all study groups, the percentage of islets with IL-1β cells in peri- and/or intra-islet regions was highly variable and ranged from 4.48% to 17.59% in group 1, 1.42% to 44.26% in group 2, 7.93% to 17.53% in group 3 and 3.85% to 42.86% in group 4, except in a single case where the value was 75%. In 25/32 donors, a higher percentage of islets showed IL-1β-positive cells in peri-islet than in intra-islet regions. In sections from diabetic donors (groups 1 and 4), a higher mean number of IL-1β-positive cells occurred in insulin-positive islets than in insulin-negative islets. In group 2, 70-90% of islets in 3/13 sections had weak-to-moderate IL-1β staining in alpha cells but staining was virtually absent or substantially reduced in the remaining groups. The mean number of exocrine IL-1β-positive cells in group 1 was lower than in the other groups. At onset of type 1 diabetes, the low number of islet-associated IL-1β-positive cells may be insufficient to elicit beta cell destruction. The variable expression in alpha cells in groups 2-4 suggests their cellular heterogeneity and probable physiological role. The significance of a higher but variable number of exocrine IL-1β-positive cells seen in non-diabetic individuals and those with long-term type 1 diabetes remains unclear.

Pancreas Islet Transplantation for Patients With Type 1 Diabetes Mellitus: A Clinical Evidence Review.

PubMed

2015-01-01

Type 1 diabetes mellitus is caused by the autoimmune destruction of pancreatic beta (β) cells, resulting in severe insulin deficiency. Islet transplantation is a β-cell replacement therapeutic option that aims to restore glycemic control in patients with type 1 diabetes. The objective of this study was to determine the clinical effectiveness of islet transplantation in patients with type 1 diabetes, with or without kidney disease. We conducted a systematic review of the literature on islet transplantation for type 1 diabetes, including relevant health technology assessments, systematic reviews, meta-analyses, and observational studies. We used a two-step process: first, we searched for systematic reviews and health technology assessments; second, we searched primary studies to update the chosen health technology assessment. The Assessment of Multiple Systematic Reviews measurement tool was used to examine the methodological quality of the systematic reviews and health technology assessments. We assessed the quality of the body of evidence and the risk of bias according to the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) Working Group criteria. Our searched yielded 1,354 citations. One health technology assessment, 11 additional observational studies to update the health technology assessment, one registry report, and four guidelines were included; the observational studies examined islet transplantation alone, islet-after-kidney transplantation, and simultaneous islet-kidney transplantation. In general, low to very low quality of evidence exists for islet transplantation in patients with type 1 diabetes with difficult-to-control blood glucose levels, with or without kidney disease, for these outcomes: health-related quality of life, secondary complications of diabetes, glycemic control, and adverse events. However, high quality of evidence exists for the specific glycemic control outcome of insulin independence compared with intensive insulin therapy. For patients without kidney disease, islet transplantation improves glycemic control and diabetic complications for patients with type 1 diabetes when compared with intensive insulin therapy. However, results for health-related quality of life outcomes were mixed, and adverse events were increased compared with intensive insulin therapy. For patients with type 1 diabetes with kidney disease, islet-after-kidney transplantation or simultaneous islet-kidney transplantation also improved glycemic control and secondary diabetic complications, although the evidence was more limited for this patient group. Compared with intensive insulin therapy, adverse events for islet-after-kidney transplantation or simultaneous islet-kidney transplantation were increased, but were in general less severe than with whole pancreas transplantation. For patients with type 1 diabetes with difficult-to-control blood glucose levels, islet transplantation may be a beneficial β-cell replacement therapy to improve glycemic control and secondary complications of diabetes. However, there is uncertainty in the estimates of effectiveness because of the generally low to very low quality of evidence for all outcomes of interest.

Islet transplantation as safe and efficacious method to restore glycemic control and to avoid severe hypoglycemia after donor organ failure in pancreas transplantation.

PubMed

Gerber, Philipp A; Hochuli, Michel; Benediktsdottir, Bara D; Zuellig, Richard A; Tschopp, Oliver; Glenck, Michael; de Rougemont, Olivier; Oberkofler, Christian; Spinas, Giatgen A; Lehmann, Roger

2018-01-01

The aim of this study was to assess safety and efficacy of islet transplantation after initial pancreas transplantation with subsequent organ failure. Patients undergoing islet transplantation at our institution after pancreas organ failure were compared to a control group of patients with pancreas graft failure, but without islet transplantation and to a group receiving pancreas retransplantation. Ten patients underwent islet transplantation after initial pancreas transplantation failed and were followed for a median of 51 months. The primary end point of HbA1c <7.0% and freedom of severe hypoglycemia was met by nine of 10 patients after follow-up after islet transplantation and in all three patients in the pancreas retransplantation group, but by none of the patients in the group without retransplantation (n = 7). Insulin requirement was reduced by 50% after islet transplantation. Kidney function (eGFR) declined with a rate of -1.0 mL ± 1.2 mL/min/1.73 m 2 per year during follow-up after islet transplantation, which tended to be slower than in the group without retransplantation (P = .07). Islet transplantation after deceased donor pancreas transplant failure is a method that can safely improve glycemic control and reduce the incidence of severe hypoglycemia and thus establish similar glycemic control as after initial pancreas transplantation, despite the need of additional exogenous insulin. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Islet transplantation using donors after cardiac death: report of the Japan Islet Transplantation Registry.

PubMed

Saito, Takuro; Gotoh, Mitsukazu; Satomi, Susumu; Uemoto, Shinji; Kenmochi, Takashi; Itoh, Toshinori; Kuroda, Yoshikazu; Yasunami, Youichi; Matsumoto, Shnichi; Teraoka, Satoshi

2010-10-15

This report summarizes outcomes of islet transplantation employing donors after cardiac death (DCD) between 2004 and 2007 as reported to the Japan Islet Transplantation Registry. Sixty-five islet isolations were performed for 34 transplantations in 18 patients with insulin-dependent diabetes mellitus, including two patients who had prior kidney transplantation. All but one donor (64/65) was DCD at the time of harvesting. Factors influencing criteria for islet release included duration of low blood pressure of the donor, cold ischemic time, and usage of Kyoto solution for preservation. Multivariate analysis selected usage of Kyoto solution as most important. Of the 18 recipients, 8, 4, and 6 recipients received 1, 2, and 3 islet infusions, respectively. Overall graft survival defined as C-peptide level more than or equal to 0.3 ng/mL was 76.5%, 47.1%, and 33.6% at 1, 2, and 3 years, respectively, whereas corresponding graft survival after multiple transplantations was 100%, 80.0%, and 57.1%, respectively. All recipients remained free of severe hypoglycemia while three achieved insulin independence for 14, 79, and 215 days. HbA1c levels and requirement of exogenous insulin were significantly improved in all patients. Islet transplantation employing DCD can ameliorate severe hypoglycemic episodes, significantly improve HbA1c levels, sustain significant levels of C-peptide, and achieve insulin independence after multiple transplantations. Thus, DCD can be an important resource for islet transplantation if used under strict releasing criteria and in multiple transplantations, particularly in countries where heart-beating donors are not readily available.

Regulatory challenges in manufacturing of pancreatic islets.

PubMed

Linetsky, E; Ricordi, C

2008-03-01

At the present time, transplantation of pancreatic islet cells is considered an experimental therapy for a selected cohort of patients with type 1 diabetes, and is conducted under an Investigational New Drug (IND) application. Encouraging results of the Edmonton Protocol published in the year 2000 sparked a renewed interest in clinical transplantation of allogeneic islets, triggering a large number of IND applications for phase I clinical trials. Promising results reported by a number of centers since then prompted the Food and Drug Administration (FDA) to consider the possibility of licensing allogeneic islets as a therapeutic treatment for patients with type 1 diabetes. However, prior to licensure, issues such as safety, purity, efficacy, and potency of the islet product must be addressed. This is complicated by the intricate nature of pancreatic islets and limited characterization prior to transplantation. In this context, control of the manufacturing process plays a critical role in the definition of the final product. Despite significant progress made in standardization of the donor organ preservation methods, reagents used, and characterization assays performed to qualify an islet cell product, control of the isolation process remains a challenge. Within the scope of the FDA regulations, islet cells meet the definition of a biologic product, somatic cell therapy, and a drug. In addition, AABB standards that address cellular therapy products apply to manufacturing facilities accredited by this organization. Control of the source material, isolation process, and final product are critical issues that must be addressed in the context of FDA and other relevant regulations applicable to islet cell products.

A preclinical evaluation of alternative site for islet allotransplantation

PubMed Central

He, Sirong; Yuan, Yujia; Han, Pengfei; Wang, Dan; Chen, Younan; Liu, Jingping; Tian, Bole; Yang, Guang; Yi, Shounan; Gao, Fabao; Zhong, Zhihui; Li, Hongxia; Cheng, Jingqiu; Lu, Yanrong

2017-01-01

The bone marrow cavity (BMC) has recently been identified as an alternative site to the liver for islet transplantation. This study aimed to compare the BMC with the liver as an islet allotransplantation site in diabetic monkeys. Diabetes was induced in Rhesus monkeys using streptozocin, and the monkeys were then divided into the following three groups: Group1 (islets transplanted in the liver with immunosuppressant), Group 2 (islets transplanted in the tibial BMC), and Group 3 (islets transplanted in the tibial BMC with immunosuppressant). The C-peptide and blood glucose levels were preoperatively measured. An intravenous glucose tolerance test (IVGTT) was conducted to assess graft function, and complete blood cell counts were performed to assess cell population changes. Cytokine expression was measured using an enzyme-linked immune sorbent assay (ELISA) and MILLIPLEX. Five monkeys in Group 3 exhibited a significantly increased insulin-independent time compared with the other groups (Group 1: 78.2 ± 19.0 days; Group 2: 58.8 ± 17.0 days; Group 3: 189.6 ± 26.2 days) and demonstrated increases in plasma C-peptide 4 months after transplantation. The infusion procedure was not associated with adverse effects. Functional islets in the BMC were observed 225 days after transplantation using the dithizone (DTZ) and insulin/glucagon stains. Our results showed that allogeneic islets transplanted in the BMC of diabetic Rhesus monkeys remained alive and functional for a longer time than those transplanted in the liver. This study was the first successful demonstration of allogeneic islet engraftment in the BMC of non-human primates (NHPs). PMID:28358858

Reversal of diabetes by pancreatic islet transplantation into a subcutaneous, neovascularized device.

PubMed

Pileggi, Antonello; Molano, R Damaris; Ricordi, Camillo; Zahr, Elsie; Collins, Jill; Valdes, Rafael; Inverardi, Luca

2006-05-15

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. 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. 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. Ease of implantation, biocompatibility, and ability to maintain long-term graft function support the potential of our implantable device for cellular-based reparative therapies.

Geometric phase transition in the cellular network of the pancreatic islets may underlie the onset of type 1diabetes

NASA Astrophysics Data System (ADS)

Wang, Xujing

Living systems are characterized by complexity in structure and emergent dynamic orders. In many aspects the onset of a chronic disease resembles phase transition in a dynamic system: quantitative changes accumulate largely unnoticed until a critical threshold is reached, which causes abrupt qualitative changes of the system. In this study we investigate this idea in a real example, the insulin-producing pancreatic islet β-cells and the onset of type 1 diabetes. Within each islet, the β-cells are electrically coupled to each other, and function as a network with synchronized actions. Using percolation theory we show how normal islet function is intrinsically linked to network connectivity, and the critical point where the islet cellular network loses site percolation, is consistent with laboratory and clinical observations of the threshold β-cell loss that causes islet functional failure. Numerical simulations confirm that the islet cellular network needs to be percolated for β-cells to synchronize. Furthermore, the interplay between site percolation and bond strength predicts the existence of a transient phase of islet functional recovery after disease onset and introduction of treatment, potentially explaining a long time mystery in the clinical study of type 1 diabetes: the honeymoon phenomenon. Based on these results, we hypothesized that the onset of T1D may be the result of a phase transition of the islet β-cell network. We further discuss the potential applications in identifying disease-driving factors, and the critical parameters that are predictive of disease onset.

Activated effector and memory T cells contribute to circulating sCD30: potential marker for islet allograft rejection.

PubMed

Saini, D; Ramachandran, S; Nataraju, A; Benshoff, N; Liu, W; Desai, N; Chapman, W; Mohanakumar, T

2008-09-01

T-cell activation up-regulates CD30 resulting in an increase in serum soluble CD30 (sCD30). CD4+ T cells, a major source for sCD30, play a significant role in the pathogenesis of rejection. In this study, sCD30 was measured pre- and posttransplant in mouse islet allograft models and human islet allograft recipients. sCD30 was measured by ELISA in diabetic C57BL/6, CD4Knockout (KO) and CD8KO islet allograft recipients. sCD30 increased significantly prior to rejection (1.8 +/- 1 days) in 80% of allograft recipients. Sensitization with donor splenocytes, or a second graft, further increased sCD30 (282.5 +/- 53.5 for the rejecting first graft vs. 374.6 +/- 129 for the rejecting second graft) prior to rejection suggesting memory CD4+ T cells contribute to sCD30. CD4KO failed to reject islet allograft and did not demonstrate sCD30 increase. CD8KO showed elevated (227 +/- 107) sCD30 (1 day) prior to rejection. High pretransplant sCD30 (>20 U/ml) correlated with poor outcome in human islet allograft recipients. Further, increase in sCD30 posttransplant preceded (3-4 months) loss of islet function. We conclude that sCD30 is released from activated CD4 T cells prior to islet allograft rejection and monitoring sCD30 can be a valuable adjunct in the follow-up of islet transplant recipients.