Islet Grafts In Mice Research Articles

Suppression of IGRP-specific CD8 T cells following induction of tolerance to a Hybrid Insulin Peptide CD4 neoepitope

Abstract An autoimmune-mediated death of insulin-producing beta cells, orchestrated by effector CD4 and CD8 T cells that recognize islet antigens, results in Type 1 Diabetes. A dominant CD4 islet autoantigen in the NOD mouse model of autoimmune diabetes is a neoepitope termed the 2.5 Hybrid Insulin Peptide (2.5HIP). By delivering the 2.5HIP as an antigen-specific immunotherapy on tolerogenic PLG-nanoparticles, islet grafts in diabetic NOD mice survived longer and cytokine production in autoreactive CD4 and CD8 T cells was suppressed, including the well-studied IGRP tetramer+ (tet+) CD8 T cells. Using both diabetic transplant recipients and prediabetic NOD mice treated with 2.5HIP nanoparticles, we examined mechanisms of peripheral tolerance induction to a dominant CD4 neoepitope and the impact on the function of IGRP CD8 T cells. Following induction of tolerance to the 2.5HIP, there was an increase in dysfunctional surface marker expression (PD1+ TIM3+) on effector 2.5HIP tet+ CD4 T cells in the spleen of treated mice. Along with a decrease in effector function, we observed an increase in the fraction of both Treg and Tr1 2.5HIP tet+ T cells expressing IL10 in the islets, spleen, and draining lymph nodes of tolerized mice. Concurrently, IGRP tet+ CD8 T cells accumulated in the draining lymph nodes and were less able to traffic and infiltrate islets. Of the IGRP tet+ cells that entered islets, there were fewer cytolytic CX 3CR1 +effector cells in tolerized mice. The robust functional increase in both Treg and Tr1 2.5HIP tet+ T cells may provide a potential mechanism for the accumulation of IGRP tet+ CD8 T cells in the draining lymph node and explain their inefficient trafficking into, and function within, islets following tolerance induction. NIH - T32 5T32DK120520-03, R01 2R01DK081166-11) JDRF 2-SRA-2020-907-S-B

Pericyte Control of Blood Flow in Intraocular Islet Grafts Impacts Glucose Homeostasis in Mice.

The pancreatic islet depends on blood supply to efficiently sense plasma glucose levels and deliver insulin and glucagon into the circulation. Long believed to be passive conduits of nutrients and hormones, islet capillaries were recently found to be densely covered with contractile pericytes with the capacity to locally control blood flow. Here, we determined the contribution of pericyte regulation of islet blood flow to plasma insulin and glucagon levels and glycemia. Selective optogenetic activation of pericytes in intraocular islet grafts contracted capillaries and diminished blood flow. In awake mice, acute light-induced stimulation of islet pericytes decreased insulin and increased glucagon plasma levels, producing hyperglycemic effects. Interestingly, pericytes are the targets of sympathetic nerves in the islet, suggesting that sympathetic control of hormone secretion may occur in part by modulating pericyte activity and blood flow. Indeed, in vivo activation of pericytes with the sympathetic agonist phenylephrine decreased blood flow in mouse islet grafts, lowered plasma insulin levels, and increased glycemia. We further show that islet pericytes and blood vessels in living human pancreas slices responded to sympathetic input. Our findings indicate that pericytes mediate vascular responses in the islet that are required for adequate hormone secretion and glucose homeostasis. Vascular and neuronal alterations that are commonly seen in the islets of people with diabetes may impair regulation of islet blood flow and thus precipitate islet dysfunction.

Inhibition of NLRC5 regulates cytokine expression in CD4+ T helper lymphocytes and prolongs murine islet and skin allograft survival

The purpose of this study was to study the effect of inhibiting NLRC5 expression and function on CD4 + T cells, and islet and skin transplantation in mice. A murine skin graft model and islet cell transplantation model were established, and the expression of NLRC5 was compared in rejection and immune tolerance groups. Mice spleen-derived CD4 + T cells were cultured, purified, and enriched in vitro, and transfected with the shRNA lentiviral vector NLRC5-RNAi-GFP. Changes in cytokine secretion were detected to understand changes in immunological function. Murine islet and skin transplantation models were injected with CD4 + T cells transfected with the lentivirus, and the survival time of the grafts and the levels of IFN-γ and IL-10 were compared between groups. The expression of NLRC5 mRNA in islet and skin grafts was significantly increased. In vitro experiments showed that the expression of IL-4 and IL-10 was up-regulated in CD4 + T cells, and T cells differentiation turned to Th2 after inhibition of NLRC5. In vivo experiments showed that inhibition of NLRC5 prolonged islet and skin graft survival. Pathological examination showed that the rejection of transplanted skin and islets in the NLRC5-RNAi group was mild, and there was a correlation between high expression of NLRC5 and rejection of mouse islet and skin grafts. In summary, inhibition of NLRC5 can prolong islet and skin graft survival induce transplant immune tolerance through induction of the secretion of Th2 cytokines by CD4 + T cells.

Recruited fibroblasts reconstitute the peri-islet membrane: a longitudinal imaging study of human islet grafting and revascularisation

Aims/hypothesisRapid and adequate islet revascularisation and restoration of the islet–extracellular matrix (ECM) interaction are significant factors influencing islet survival and function of the transplanted islets in individuals with type 1 diabetes. Because the ECM encapsulating the islets is degraded during islet isolation, understanding the process of revascularisation and engraftment after transplantation is essential and needs further investigation.MethodsHere we apply a longitudinal and high-resolution imaging approach to investigate the dynamics of the pancreatic islet engraftment process up to 11 months after transplantation. Human and mouse islet grafts were inserted into the anterior chamber of the mouse eye, using a NOD.ROSA-tomato.Rag2−/− or B6.ROSA-tomato host allowing the investigation of the expansion of host vs donor cells and the contribution of host cells to aspects such as promoting the encapsulation and vascularisation of the graft.ResultsA fibroblast-like stromal cell population of host origin rapidly migrates to ensheath the transplanted islet and aid in the formation of a basement membrane-like structure. Moreover, we show that the vessel network, while reconstituted by host endothelial cells, still retains the overall architecture of the donor islets.Conclusions/interpretationIn this transplantation situation the fibroblast-like stromal cells appear to take over as main producers of ECM or act as a scaffold for other ECM-producing cells to reconstitute a peri-islet-like basement membrane. This may have implications for our understanding of long-term graft rejection and for the design of novel strategies to interfere with this process.

Circulating miRNA-150-5p is associated with immune-mediated early β-cell loss in a humanized mouse model.

Aberrant microRNA (miRNA) expression levels are associated with various graft rejections. We used our humanized mouse model with transplanted human islets to identify miRNAs in islet grafts related to xenograft rejection and circulating miRNAs associated with xenograft rejection-mediated β-cell loss. Diabetic immunodeficient NOD.scid mice were transplanted with human islets and subsequently achieved stable normoglycemia. Lymphocytes from NOD mice were then adoptively transferred to the humanized mice to induce human β-cell destruction. Islet graft and plasma were collected immediately once blood glucose reached >200mg/dL. miRNAs in the islet grafts and in the plasma with or without adoptive lymphocyte transfer (ALT) were measured using NanoString nCounter® miRNA Expression Assay and qPCR. A set of immune-related miRNAs was significantly increased in human islet grafts of ALT-treated mice compared to control mice. Of these miRNAs, miR-150-5p was significantly increased in the circulation of ALT-treated mice at tissue collection and the increase was a result of immune activation rather than simply the presence of lymphocytes in circulation. Furthermore, miR-150-5p was significantly increased in human islet graft and circulation prior to the development of hyperglycemia in the ALT-treated mice. Our data demonstrated that immune-related miRNAs are associated with human islet xenograft rejection in mice. miR-150-5p is increased in human islet graft and in the circulation during islet xenograft rejection and β-cell destruction prior to hyperglycemia and may be an early biomarker for islet xenograft rejection.

Survival and Metabolic Function of Syngeneic Mouse Islet Grafts Transplanted Into the Hepatic Sinus Tract.

Islet grafts are transplanted into the liver via a portal vein in 90% of the clinical islet transplantations. However, the portal vein is far from being the ideal infusion site due to its unique drawbacks. These issues necessitated the exploration of an alternatively optimized site for clinical islet transplantation. With the widespread clinical application of percutaneous transhepatic puncture technique, we envisioned the possibility of islet transplantation into the hepatic sinus tract (HST). The HST was created by temporarily placing a medically approved material into the hepatic parenchyma of C57BL/6 mice. The syngeneic islets were transplanted into the HST, after which, the nonfasting blood glucose, intraperitoneal glucose tolerance, and morphology were evaluated. A collagen-lined HST was formed by the 28-day implantation of a cylindrical nylon rod. Transplantation of ~300 syngeneic islets into the HST routinely reversed the hyperglycemia of the recipient mice and maintained normoglycemia for longer than 100 days until the graft was removed. The islet grafts within the HST stained positively for insulin, glucagon, and abundant microvessels and achieved comparable results to the islet grafts under the kidney capsule with respect to glycemic control and glucose tolerance. These results suggested that an HST can be constructed for islet transplantation by temporarily placing a nylon material in the liver parenchyma. The HST is a promising site for clinical islet transplantation, thereby providing a satisfactory environment for the survival and metabolic function of islet grafts.

Syntaxin 4 Expression in Pancreatic β-Cells Promotes Islet Function and Protects Functional β-Cell Mass.

Syntaxin 4 (Stx4) enrichment in human and mouse islet grafts improves the success of transplants in reversing streptozotocin (STZ)-induced diabetes in mice, although the underlying molecular mechanisms remain elusive. Toward a further understanding of this, human islets and inducible transgenic mice that selectively overexpress Stx4 in islet β-cells (βTG-Stx4) were challenged with proinflammatory stressors in vitro and in vivo. Remarkably, βTG-Stx4 mice resisted the loss of β-cell mass and the glucose intolerance that multiple low doses of STZ induce. Under standard conditions, glucose tolerance was enhanced and mice maintained normal fasting glycemia and insulinemia. Conversely, Stx4 heterozygous knockout mice succumbed rapidly to STZ-induced glucose intolerance compared with their wild-type littermates. Human islet β-cells overexpressing Stx4 exhibited enhanced insulin secretory capability; resilience against proinflammatory cytokine–induced apoptosis; and reduced expression of the CXCL9, CXCL10, and CXCL11 genes coordinate with decreased activation/nuclear localization of nuclear factor-κB. Finding ways to boost Stx4 expression presents a novel potential therapeutic avenue for promoting islet function and preserving β-cell mass.

A Model for Human Islet Transplantation to Immunodeficient Streptozotocin-Induced Diabetic Mice

Streptozotocin (STZ) is a cytotoxic glucose analogue that causes beta cell death and is widely used to induce experimental diabetes in rodents. The sensitivity of beta cells to STZ is species-specific and human beta cells are resistant to STZ. In experimental islet transplantation to rodents, STZ-diabetes must be induced before transplantation to avoid destruction of grafted islets by STZ. In human islet transplantation, injection of STZ before transplantation is inconvenient and costly, since human islet availability depends on organ donation and frail STZ-diabetic mice must be kept for unpredictable lapses of time until a human islet preparation is available. Based on the high resistance of human beta cells to STZ, we have tested a new model for STZ-diabetes induction in which STZ is injected after human islet transplantation. Human and mouse islets were transplanted under the kidney capsule of athymic nude mice, and 10–14 days after transplantation mice were intraperitoneally injected with five consecutive daily doses of STZ or vehicle. Beta-cell death increased and beta-cell mass was reduced in mouse islet grafts after STZ injection. In contrast, in human islet grafts beta cell death and mass did not change after STZ injection. Mice transplanted with rodent islets developed hyperglycemia after STZ-injection. Mice transplanted with human islets remained normoglycemic and developed hyperglycemia when the graft was harvested. STZ had no detectable toxic effects on beta cell death, mass and function of human transplanted islets. We provide a new, more convenient and cost-saving model for human islet transplantation to STZ-diabetic recipients in which STZ is injected after islet transplantation.

Local immunomodulation Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance.

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 receptor following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of Fas ligand (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.

Vegf-A mRNA transfection as a novel approach to improve mouse and human islet graft revascularisation.

The initial avascular period following islet transplantation seriously compromises graft function and survival. Enhancing graft revascularisation to improve engraftment has been attempted through virus-based delivery of angiogenic triggers, but risks associated with viral vectors have hampered clinical translation. In vitro transcribed mRNA transfection circumvents these risks and may be used for improving islet engraftment. Mouse and human pancreatic islet cells were transfected with mRNA encoding the angiogenic growth factor vascular endothelial growth factor A (VEGF-A) before transplantation under the kidney capsule in mice. At day 7 post transplantation, revascularisation of grafts transfected with Vegf-A (also known as Vegfa) mRNA was significantly higher compared with non-transfected or Gfp mRNA-transfected controls in mouse islet grafts (2.11- and 1.87-fold, respectively) (vessel area/graft area, mean ± SEM: 0.118 ± 0.01 [n = 3] in Vegf-A mRNA transfected group (VEGF) vs 0.056 ± 0.01 [n = 3] in no RNA [p < 0.05] vs 0.063 ± 0.02 [n = 4] in Gfp mRNA transfected group (GFP) [p < 0.05]); EndoC-bH3 grafts (2.85- and 2.48-fold. respectively) (0.085 ± 0.02 [n = 4] in VEGF vs 0.030 ± 0.004 [n = 4] in no RNA [p < 0.05] vs 0.034 ± 0.01 [n = 5] in GFP [p < 0.05]); and human islet grafts (3.17- and 3.80-fold, respectively) (0.048 ± 0.013 [n = 3] in VEGF vs 0.015 ± 0.0051 [n = 4] in no RNA [p < 0.01] vs 0.013 ± 0.0046 [n = 4] in GFP [p < 0.01]). At day 30 post transplantation, human islet grafts maintained a vascularisation benefit (1.70- and 1.82-fold, respectively) (0.049 ± 0.0042 [n = 8] in VEGF vs 0.029 ± 0.0052 [n = 5] in no RNA [p < 0.05] vs 0.027 ± 0.0056 [n = 4] in GFP [p < 0.05]) and a higher beta cell volume (1.64- and 2.26-fold, respectively) (0.0292 ± 0.0032 μl [n = 7] in VEGF vs 0.0178 ± 0.0021 μl [n = 5] in no RNA [p < 0.01] vs 0.0129 ± 0.0012 μl [n = 4] in GFP [p < 0.001]). Vegf-A mRNA transfection before transplantation provides a promising and safe strategy to improve engraftment of islets and other cell-based implants.

Carbon Monoxide Inhibits Islet Apoptosis via Induction of Autophagy.

Carbon monoxide (CO) functions as a therapeutic molecule in various disease models because of its anti-inflammatory and antiapoptotic properties. We investigated the capacity of CO to reduce hypoxia-induced islet cell death and dysfunction in human and mouse models. Culturing islets in CO-saturated medium protected them from hypoxia-induced apoptosis and preserved β cell function by suppressing expression of proapoptotic (Bim, PARP, Cas-3), proinflammatory (TNF-α), and endoplasmic reticulum (ER) stress (glucose-regulated protein 94, grp94, CHOP) proteins. The prosurvival effects of CO on islets were attenuated when autophagy was blocked by specific inhibitors or when either ATG7 or ATG16L1, two essential factors for autophagy, was downregulated by siRNA. In vivo, CO exposure reduced both inflammation and cell death in grafts immediately after transplantation, and enhanced long-term graft survival of CO-treated human and mouse islet grafts in streptozotocin-induced diabetic non-obese diabetic severe combined immunodeficiency (NOD-SCID) or C57BL/6 recipients. These findings underline that pretreatment with CO protects islets from hypoxia and stress-induced cell death via upregulation of ATG16L1-mediated autophagy. Our results suggested that CO exposure may provide an effective means to enhance survival of grafts in clinical islet cell transplantation, and may be beneficial in other diseases in which inflammation and cell death pose impediments to achieving optimal therapeutic effects. Antioxid. Redox Signal. 28, 1309-1322.

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

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

Islet-derived damage-associated molecular pattern molecule contributes to immune responses following microencapsulated neonatal porcine islet xenotransplantation inmice.

Clinical allogeneic islet transplantation has become an attractive procedure for type 1 diabetes mellitus treatment. However, there is a severe shortage of human donors. Microencapsulated neonatal porcine islet (NPI) xenotransplantation may be an alternative transplantation procedure. Currently, the efficacy of microencapsulated NPI xenotransplantation into the peritoneal cavity is limited because of early non-function resulting from inflammation, which is a serious hindrance to promoting this procedure as a standard therapy. Previously, we have demonstrated that high-mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, was released from transplanted islets and triggered inflammatory reactions leading to early loss of intrahepatic syngeneic islet grafts in mice. In this study, we hypothesized that the inflammatory reaction in the peritoneal cavity following the transplantation of microencapsulated NPIs is more severe than that of empty capsules. Additionally, we predicted that HMGB1 released from transplanted microencapsulated NPIs triggers further inflammatory reactions in mice. Finally, we hypothesized that microencapsulated NPI xenotransplantation efficacy would be improved by treatment-targeting inflammatory reactions in a mouse model. A total of 10000 empty capsules (alginate-poly-L-ornithine-alginate) or 10000 IEQ microencapsulated NPIs were transplanted into the peritoneal cavity of streptozotocin-induced diabetic C57BL/6 mice. The numbers of mononuclear cells in the peritoneal cavity following empty capsule or microencapsulated NPI transplantation were 4.8×10(6) ±0.9×10(6) and 13.6×10(6) ±3.0×10(6) , respectively (P<0.05). Fluorescence-activated cell sorting (FACS) analysis revealed that tumor necrosis factor (TNF)-α-, interleukin (IL)-6-, interferon (IFN)-γ-, and/or IL-12-positive macrophages, neutrophils, and dendritic cells had infiltrated the peritoneal cavity after empty capsules or microencapsulated NPIs administration. IL-6 concentrations in the peritoneal lavage fluids on 7days after empty capsule or microencapsulated NPI transplantation were 18.5±10.0 and 157.4±46.3pg/ml, respectively (P<0.001), while TNF-α concentrations were 4.6±1.4 and 19.8±8.4pg/ml, respectively (P<0.01). In addition, HMGB1 concentrations were 37.6±6.6 and 117.4±8.1ng/ml, respectively (P<0.0001). In vitro experiments revealed that the total amount of released HMGB1 into the culture medium of empty capsule (200 capsules/dish) and microencapsulated NPI (200 IEQ/dish) after hypoxic culture (1% O2 , 5% CO2 , and 94% N2 ) was 0 and 8.6±2.2ng, respectively (P<0.001). FACS analysis revealed that TNF-α- and IL-6-positive macrophages were also observed in the peritoneal cavity following intraperitoneal injection of HMGB1 itself. Anti-TNF-α antibody treatment was associated with slightly prolonged graft survival and improved glucose tolerance 30days after transplantation, but none of the recipients were remained normoglycemic. In conclusion, early inflammatory reactions might be therapeutic targets for the prolongation of microencapsulated NPIs graft survival. Thus, treatment-targeting inflammation might improve the efficiency of clinical microencapsulated NPI xenotransplantation.

Porcine Islet-Specific Tolerance Induced by the Combination of Anti-LFA-1 and Anti-CD154 mAbs Is Dependent on PD-1.

We previously demonstrated that short-term administration of a combination of anti-LFA-1 and anti-CD154 monoclonal antibodies (mAbs) induces tolerance to neonatal porcine islet (NPI) xenografts that is mediated by regulatory T cells (Tregs) in B6 mice. In this study, we examined whether the coinhibitory molecule PD-1 is required for the induction and maintenance of tolerance to NPI xenografts. We also determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or xenogeneic rat islet grafts since we previously demonstrated that tolerance to NPI xenografts could be extended to second-party NPI xenografts. Finally, we determined whether tolerance to NPI xenografts could be extended to allogeneic mouse or second-party porcine skin grafts. Diabetic B6 mice were transplanted with 2,000 NPIs under the kidney capsule and treated with short-term administration of a combination of anti-LFA-1 and anti-CD154 mAbs. Some of these mice were also treated simultaneously with anti-PD-1 mAb at >150 days posttransplantation. Spleen cells from some of the tolerant B6 mice were used for proliferation assays or were injected into B6 rag(-/-) mice with established islet grafts from allogeneic or xenogeneic donors. All B6 mice treated with anti-LFA-1 and anti-CD154 mAbs achieved and maintained normoglycemia until the end of the study; however, some mice that were treated with anti-PD-1 mAb became diabetic. All B6 rag(-/-) mouse recipients of first- and second-party NPIs maintained normoglycemia after reconstitution with spleen cells from tolerant B6 mice, while all B6 rag(-/-) mouse recipients of allogeneic mouse or xenogeneic rat islets rejected their grafts after cell reconstitution. Tolerant B6 mice rejected their allogeneic mouse or xenogeneic second-party porcine skin grafts while remaining normoglycemic until the end of the study. These results show that porcine islet-specific tolerance is dependent on PD-1, which could not be extended to skin grafts.

Immunotoxicological effects of streptozotocin and alloxan: In vitro and in vivo studies

Streptozotocin (STZ) and alloxan (ALX), widely used to induce diabetes in experimental animals, have different structures and mechanisms of action. We investigated those effects of these drugs on the immune system that might influence engraftment efficiency and graft survival in transplantation models, and their cytotoxicity on hematopoietic cell lines.We used the minimum dose to induce diabetes in a mouse, i.e. 180mg/kg i.v. STZ and 75mg/kg i.v. ALX. Both groups exhibited significant decrease in body weight during 4 days post-treatment as compared to controls. We found that blood glucose in ALX-injected mice increased faster than in STZ-injected mice. The total number of recovered splenocytes was lower in STZ-injected animals than in ALX-injected animals. The survival periods of rat islet grafts in recipient mice were longer and more diverse in STZ-injected recipients (7–24 days) compared to ALX-injected recipients (6–7 days). The in vitro study showed that ALX was less cytotoxic in cell lines with IC50 values of 2809, 3679 and >4000μg/ml for HL60, K562 and C1498 cells respectively. STZ was more toxic, especially in HL60 cells, with IC50 values of 11.7, 904 and 1024μg/ml for HL60, K562 and C1498 cells respectively. Furthermore, in response to concanavalin A (Con-A), splenocytes from STZ-injected mice produced higher amounts of interferon-gamma (IFN-γ) than those from ALX-injected mice. In conclusion, STZ was more cytotoxic than ALX in vitro and in vivo. STZ caused lymphocytopenia, which may result in longer graft survival in STZ-treated animals than in ALX-treated animals.

The use of hydrogen gas clearance for blood flow measurements in single endogenous and transplanted pancreatic islets

The blood perfusion of pancreatic islets is regulated independently from that of the exocrine pancreas, and is of importance for multiple aspects of normal islet function, and probably also during impaired glucose tolerance. Single islet blood flow has been difficult to evaluate due to technical limitations. We therefore adapted a hydrogen gas washout technique using microelectrodes to allow such measurements. Platinum micro-electrodes monitored hydrogen gas clearance from individual endogenous and transplanted islets in the pancreas of male Lewis rats and in human and mouse islets implanted under the renal capsule of male athymic mice. Both in the rat endogenous pancreatic islets as well as in the intra-pancreatically transplanted islets, the vascular conductance and blood flow values displayed a highly heterogeneous distribution, varying by factors 6–10 within the same pancreas. The blood flow of human and mouse islet grafts transplanted in athymic mice was approximately 30% lower than that in the surrounding renal parenchyma. The present technique provides unique opportunities to study the islet vascular dysfunction seen after transplantation, but also allows for investigating the effects of genetic and environmental perturbations on islet blood flow at the single islet level in vivo.

Three-dimensional islet graft histology: panoramic imaging of neural plasticity in sympathetic reinnervation of transplanted islets under the kidney capsule

Microscopic examination of transplanted islets in an ectopic environment provides information to evaluate islet engraftment, including revascularization and reinnervation. However, because of the dispersed nature of blood vessels and nerves, global visualization of the graft neurovascular network has been difficult. In this research we revealed the neurovascular network by preparing transparent mouse islet grafts under the kidney capsule with optical clearing to investigate the sympathetic reinnervation via three-dimensional confocal microscopy. Normoglycemic and streptozotocin-induced diabetic mice were used in syngeneic islet transplantation, with both groups maintaining euglycemia after transplantation. Triple staining of insulin/glucagon, blood vessels, and tyrosine hydroxylase (sympathetic marker) was used to reveal the graft microstructure, vasculature, and sympathetic innervation. Three weeks after transplantation, we observed perigraft sympathetic innervation similar to the peri-islet sympathetic innervation in the pancreas. Six weeks after transplantation, prominent intragraft, perivascular sympathetic innervation was achieved, resembling the pancreatic intraislet, perivascular sympathetic innervation in situ. Meanwhile, in diabetic recipients, a higher graft sympathetic nerve density was found compared with grafts in normoglycemic recipients, indicating the graft neural plasticity in response to the physiological difference of the recipients and the resolving power of this imaging approach. Overall, this new graft imaging method provides a useful tool to identify the islet neurovascular complex in an ectopic environment to study islet engraftment.

The graft survival protection of subcutaneous allogeneic islets with hydrogel grafting and encapsulated by CTLA4Ig and IL1ra

In this study, we examined the combined effect of two recombinant immunomodulator proteins, cytotoxic T-lymphocyte antigen-4-Ig (CTLA4Ig) and interleukin-1 receptor antagonist (IL1ra), on the survival of subcutaneous mouse islet grafts with temperature-sensitive hydrogel, poly(N-isopropylacrylamide) (pNIPAAm)-chitosan-hyaluronic acid (CPNHA), and gelatin microspheres in an allogeneic mouse model. The phase-transition temperature for the CTLA4Ig-grafted CPNHA (CPNCHA) was 28 °C. An in vitro cytotoxicity assay and in vivo tissue reactions showed that both CPNCHA and the gelatin microspheres were biocompatible, biodegradable and nontoxic. The survival of the allogeneic islets was prolonged when the grafts were transplanted into a subcutaneous matrix composed of CPNCHA and gelatin microspheres containing IL1ra relative to those implanted in CPNHA containing gelatin microspheres without an immunomodulator. In conclusion, our results suggest that the temperature-sensitive hydrogel, CPNCHA, is a suitable subcutaneous matrix for islet transplantation, and grafted CTLA4Ig and encapsulated IL1ra reduce immune rejection and prolong the survival of the functioning allogeneic islets. The combined effect of cytotoxic T-lymphocyte antigen-4-Ig (CTLA4Ig) and interleukin-1 receptor antagonist (IL1ra) on the survival of subcutaneous mouse islet grafts with temperature-sensitive hydrogel, pNIPAAm-chitosan-hyaluronic acid (CPNHA), and gelatin microspheres in an allogeneic mouse model is examined. The survival of the allogeneic islets was prolonged when the grafts were transplanted into a subcutaneous matrix composed of CPN-CTLA4Ig-HA (CPNCHA) and gelatin microspheres containing IL1ra relative to those implanted in CPNHA containing gelatin microspheres without an immunomodulator.

Magnetic Resonance Imaging of Mouse Islet Grafts Labeled with Novel Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles

ObjectTo better understand the fate of islet isografts and allografts, we utilized a magnetic resonance (MR) imaging technique to monitor mouse islets labeled with a novel MR contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles.Materials and MethodsAfter being incubated with and without CSPIO (10 µg/ml), C57BL/6 mouse islets were examined under transmission electron microscope (TEM) and their insulin secretion was measured. Cytotoxicity was examined in α (αTC1) and β (NIT-1 and βTC) cell lines as well as islets. C57BL/6 mice were used as donors and inbred C57BL/6 and Balb/c mice were used as recipients of islet transplantation. Three hundred islets were transplanted under the left kidney capsule of each mouse and then MR was performed in the recipients periodically. At the end of study, the islet graft was removed for histology and TEM studies.ResultsAfter incubation of mouse islets with CSPIO (10 µg/mL), TEM showed CSPIO in endocytotic vesicles of α- and β-cells at 8 h. Incubation with CSPIO did not affect insulin secretion from islets and death rates of αTC1, NIT-1 and βTC cell lines as well as islets. After syngeneic and allogeneic transplantation, grafts of CSPIO-labeled islets were visualized on MR scans as persistent hypointense areas. At 8 weeks after syngeneic transplantation and 31 days after allogeneic transplantation, histology of CSPIO-labeled islet grafts showed colocalized insulin and iron staining in the same areas but the size of allografts decreased with time. TEM with elementary iron mapping demonstrated CSPIO distributed in the cytoplasm of islet cells, which maintained intact ultrastructure.ConclusionOur results indicate that after syngeneic and allogeneic transplantation, islets labeled with CSPIO nanoparticles can be effectively and safely imaged by MR.

In Vivo Imaging of Transplanted Islets Labeled with a Novel Cationic Nanoparticle

To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a β-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients.