Isolation and transplantation of rodent islets are frequently used as a tool for predicting the behavior of new protocols for islet allotransplants in type 1 diabetes patients. Bovine serum albumin (BSA) is recognized as a protease inhibitor possibly protecting function and viability in islets. For this study, the addition of 0.2% BSA to the isolation protocol resulted in a 30% increase in islet yields while other parameters, such as viability and function, retained high islet quality. In vivo, a minimal mass of 70 BSA treated islets showed their ability to control glycemia levels in diabetic mice by bringing the average blood glucose to 153 ± 13.2 mg/dL compared to 288 ± 22.6 mg/dL without BSA. Our results show that the simple addition of BSA to the isolation protocol constitutes a reliable and reproducible method for increasing islet yield. Also adding BSA to the transplantation medium improves islet function in vivo. The method outlined here can reduce the overall number of animals needed per experiment and also reduce the time and resources needed for islet preparation.

Orthotopic liver transplantation was carried out in baboons using wild-type (WT, n = 1) or genetically-engineered pigs (alpha 1,3-galactosyltransferase gene-knockout, GTKO), n = 1; GTKO pigs transgenic for human CD46, n = 7) and a clinically-acceptable immunosuppressive regimen. Biopsies were obtained from the WT pig liver pre-Tx and at 30 min, 1, 2, 3, 4 and 5 h post-transplantation. Biopsies of genetically-engineered livers were obtained pre-Tx, 2 h after reperfusion and at necropsy (4-7 days after transplantation). Tissues were examined by light, confocal, and electron microscopy. All major native organs were also examined. The WT pig liver underwent hyperacute rejection. After genetically-engineered pig liver transplantation, hyperacute rejection did not occur. Survival was limited to 4-7 days due to repeated spontaneous bleeding in the liver and native organs (as a result of profound thrombocytopenia) which necessitated euthanasia. At 2 h, graft histology was largely normal. At necropsy, genetically-engineered pig livers showed hemorrhagic necrosis, platelet aggregation, platelet-fibrin thrombi, monocyte/macrophage margination mainly in liver sinusoids, and vascular endothelial cell hypertrophy, confirmed by confocal and electron microscopy. Immunohistochemistry showed minimal deposition of IgM, and almost absence of IgG, C3, C4d, C5b-9, and of a cellular infiltrate, suggesting that neither antibody-nor cell-mediated rejection played a major role.

Anti-insulin autoimmunity is one of the primary forces in initiating and progressing β-cell destruction in type 1 diabetes. While insulin expression in thymic medullary epithelial cells has been shown to be essential for establishing β-cell central tolerance, the function of insulin expression in antigen-presenting cells (APCs) of hematopoietic lineage remains elusive. With a Cre-lox reporter approach, we labeled Aire-expressing cells with enhanced yellow fluorescent proteins, and found that insulin expression in the spleen was restricted predominantly to a population of Aire(+)CD11c(int)B220(+) dendritic cells (DCs). Targeted insulin deletion in APCs failed to induce anti-islet autoimmunity in B6 mice. In contrast, elevated levels of T cell infiltration into islets were observed in B6(g7) congenic mice when insulin was specifically deleted in their CD11c-expressing DCs (B6(g7)·CD11c-ΔIns mice). Thus, insulin expression in BM-derived, Aire(+) tolerogenic DCs may play an essential role to prevent the activation and expansion of insulin-reactive T cells in the periphery.

Currently, islet transplantation as a cell therapeutic option for type 1 diabetes occurs via islet injection into the portal vein. Direct contact between islets and blood is a pathophysiological “provocation” that results in the instant blood-mediated inflammatory reaction (IBMIR) and is associated with early islet loss. However, the nature of the various insults on the islets in the blood stream remains mostly unknown. To gain insight into the mechanisms, we utilized a simplified in vitro model in which islets were exposed to blood in different clinically relevant but increasingly challenging, autologous, allogeneic, and xenogeneic combinations. Irrespective of the blood type and species compatibility, islets triggered blood clotting. Islet damage was worse as islet, and blood compatibility diminished, with substantial islet injury after exposure of porcine islets to human blood. Islet damage involved membrane leakage, antibody deposition, complement activation, positive staining for the membrane attack complex, and mitochondrial dysfunction. Islet damage occurred even after exposure to plasma only, and specific complement inactivation and neutralization of IgM substantially prevented islet damage, indicating the importance of humoral immunity. Efficacious measures are needed to reduce this injury, especially in view of a potential clinical use of porcine islets to treat diabetes.

Previous studies by our group, using an experimental autoimmune thyroiditis (EAT) model in Strain 13 inbred guinea pigs, resulted in T cell-mediated delayed hypersensitivity; however, autoantibodies proved not to be cytotoxic to thyroid epithelial cells in the presence or absence of complement proteins. Albeit, T cell-mediated lymphocyte cytotoxicity began to diminish sharply concomitantly with increasing titers of circulating autoantibodies, indicating a skewing of the self-reactive response and amelioration of the EAT. Furthermore, immunization of guinea pigs with thyroglobulin in incomplete Freund's adjuvant (IFA) generated a high titer of antithyroglobulin antibodies and proved to inhibit thyroiditis. These observations indicated that the shift in the immune response from Th1 to Th2 and the production of antibodies were likely responsible for ameliorating EAT. Based upon these results, we extrapolated our studies to design a multivalent vaccine, which shows promise in preventing/reversing T1D in NOD mice. A small pilot study was conducted in which a total of 34 mice, 20 non-immunized controls and 14 immunized with syngeneic islet lysate, were monitored for mean day to diabetes for a total of 28 weeks. Immunization of NOD animals with syngeneic islet lysates resulted in a significant delay in diabetes onset (P < 0.001) as compared to non-immunized controls. To further assess the vaccine's efficacy, robustness, and delay of disease, a large-scale experiment was conducted and monitored for 32 weeks using 106 mice, 64 non-immunized controls and 42 immunized with syngeneic islet lysate. At the end of the study, 90% of the non-immunized group developed diabetes, while less than 25% of the immunized group became diabetic (P < 0.0001). The protective effect, as a result of vaccination, correlated with an increase in the levels of IL-10 and IL-4 cytokines as well as a skewing to Th2-dependent isotype antibodies in serum. Strikingly, adoptive transfer of spleen cells from immunized animals into NOD.scid recipients provided protection against transfer of diabetes by diabetogenic spleen cells. The results of this study provide evidence that vaccination with islet lysate leads to a Th2-dependent skewing of the immune response to islet beta cells as a possible mechanism of protection. This strategy may be implemented as a possible vaccination protocol for arresting and/or preventing T1D in patients.

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

Background: Xenotransplantation of porcine islets could be a valuable alternative to the shortage of human islets for transplantation. To overcome the immunological obstacle of antibody-mediated rejection, pigs homozygous for alpha 1,3-galactosyltransferase gene knock-out (GT-KO) have been produced. The effect of this mutation on glucose metabolism is unknown.Methods: Glucose, insulin, C-peptide and glucagon levels were studied in eight adult pigs (four wild-type [WT] and four GT-KO) during intravenous glucose tolerance test (IVGTT), arginine stimulation test (AST), and insulin tolerance test (ITT). Morphological analysis of the pancreata was also performed. The in vitro insulin response to a high glucose concentration and theophylline were studied in a dynamic perfusion system with isolated islets.Results: Basal and stimulated blood glucose levels were similar in WT and GT-KO pigs. Basal insulin, C-peptide and glucagon were higher in GT-KO pigs. C-peptide and insulin responses to arginine and glucose were also higher in GT-KO animals. The reduction in blood glucose during ITT and IVGTT was similar in WT and GT-KO pigs. The extent of staining for insulin and glucagon in the pancreata were similar. The basal insulin secretion of isolated islets was higher in GT-KO pigs, while stimulation indexes for glucose and theophylline were similar to WT.Conclusions: GT-KO pigs demonstrated differences in glucose metabolism compared to WT pigs, the cause for which remains uncertain. It is unlikely that these differences would in any way affect the outcome of GT-KO porcine islet xenotransplantation.

OBJECTIVE Because of reduced antioxidant defenses, β-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant. RESEARCH DESIGN AND METHODS The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation. RESULTS Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant–treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection. CONCLUSIONS Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic beta-cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire-expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the beta-cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID-TEC mice for insulin-deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. beta-cell-specific autoimmune destruction was observed, as well as islet-specific T cell infiltration. The presence of insulin-specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti-insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes-resistant HLA class II alleles. ID-TEC mice could serve as a new model for studying this pathology.

OBJECTIVE—To describe the ability of nonhuman primate endocrine pancreata to reestablish endogenous insulin production after chemical β-cell destruction. RESEARCH DESIGN AND METHODS—Eleven monkeys (Macaca fascicularis) were rendered diabetic with streptozotocin. Eight diabetic monkeys received intraportal porcine islet transplantation. RESULTS—Two monkeys transplanted after 75 days of type 1 diabetes showed recovery of endogenous C-peptide production a few weeks after transplantation, concomitant with graft failure. Histological analysis of the pancreas of these monkeys showed insulin-positive cells, single or in small aggregates, scattered in the pancreas and adjacent to ducts. Interestingly, numerous CK19+ cells costained with proinsulin and PDX-1 antibodies. Furthermore, the peculiar double phenotype glucagon-positive/GLUT2+ was observed. In these monkeys as well as in all others, the original islets showed no insulin staining. CONCLUSIONS—Our data provide evidence that, in nonhuman primates, the pancreas can reestablish endogenous insulin production after chemical β-cell destruction. This seems to be a nongeneralizable event with only 2 out of 11 monkeys recovering β-cell function. In these two monkeys, younger age and islet graft behavior might have played a role in triggering endogenous β-cell recovery.

Excessive proliferation of vascular smooth muscle cells (VSMCs) is one of the primary lesions in atherosclerosis development during diabetes. High glucose triggers VSMC proliferation and initiates activation of the transcription factor nuclear factor (NF)-kappaB. Recently, clinical studies have demonstrated that replacement therapy with C-peptide, a cleavage product of insulin, to type 1 diabetic (T1D) patients is beneficial on a variety of diabetes-associated vascular complications. However, the mechanisms underlying the beneficial activity of C-peptide on the vasculature in conditions of hyperglycemia are largely unknown. The effects of C-peptide on the proliferation of human umbilical artery smooth muscle cell (UASMC) and aortic smooth muscle cell (AoSMC) lines cultured under high glucose for 48 h were tested. To gain insights on potential intracellular signaling pathways affected by C-peptide, we analyzed NF-kappaB activation in VSMCs since this pathway represents a key mechanism for the accelerated vascular disease observed in diabetes. High glucose conditions (25 mmol/L) stimulated NF-kappaB-dependent VSMC proliferation since the addition of two NF-kappaB-specific inhibitors, BAY11-7082 and PDTC, prevented proliferation. C-peptide at the physiological concentrations of 0.5 and 1 nmol/L decreased high glucose-induced proliferation of VSMCs that was accompanied by decreased phosphorylation of IkappaB and reduced NF-kappaB nuclear translocation. These results suggest that in conditions of hyperglycemia C-peptide reduces proliferation of VSMCs and NF-kappaB nuclear translocation. In patients with T1D, physiological C-peptide levels may exert beneficial effects on the vasculature that, under high glucose conditions, is subject to progressive dysfunction.