Diazoxide Preconditioning of Nonhuman Primate Pancreas Improves Islet Isolation Outcomes by Mitochondrial Protection.

OBJECTIVES: Previously, we showed that diazoxide (DZ), an effective ischemic preconditioning agent, protected rodent pancreas against ischemia-reperfusion injury. Here, we further investigate whether DZ supplementation to University of Wisconsin (UW) solution during pancreas procurement and islet isolation has similar cytoprotection in a preclinical nonhuman primate model. METHODS: Cynomolgus monkey pancreata were flushed with UW or UW + 150 µM DZ during procurement and preserved for 8 hours before islet isolation. RESULTS: First, a significantly higher islet yield was observed in UW + DZ than in UW (57,887 vs 23,574 IEq/pancreas and 5396 vs 1646 IEq/g). Second, the DZ treated islets had significantly lower apoptotic cells per islet (1.64% vs 9.85%). Third, DZ significantly inhibited ROS surge during reperfusion with a dose-response manner. Fourth, DZ improved in vitro function of isolated islets determined by mitochondrial potentials and calcium influx in responses to glucose and KCI. Fifth, the DZ treated islets had much higher cure rate and better glycemia control in diabetic mice transplant model. CONCLUSIONS: This study showed a strong mitochondrial protection of DZ on nonhuman primate islets against ischemia-reperfusion injury that provides strong evidence for its clinical application in islet and pancreas transplantation.

Selective Vagotomy Worsens Glucose Control After Ileal Transposition.

PURPOSES: Our aim was to investigate the effects of selective celiac branch vagotomy on food intake and glycemic control after ileal transposition (IT) and the possible roles of the vagus on the improvement of diabetes. MATERIALS AND METHODS: Forty non-obese rats with diabetes underwent either IT, IT + celiac branch vagotomy (ITV), sham IT (SI), or sham IT + celiac branch vagotomy (SIV). They were pair fed, and the food intake, body weight, fasting plasma glucose, and glucagon-like peptide 1 (GLP-1) level were monitored. The number of activated pro-opiomelanocortin (POMC) neurons and POMC-derived peptides were measured after sacrifice. RESULTS: The fasting glucose level of the ITV group was higher (7.0 ± 0.7 mmol/L vs. 5.7 ± 0.3, P = 0.01), and the area under the curve of the oral glucose tolerance test (AUCOGTT) value was greater than that of the IT group (1101.8 ± 90.3 (mmol/l) min vs. 986.9 ± 47.7 (mmol/l) min, P = 0.01). There was no significant difference in the postprandial GLP-1 level between these two groups, but the number of activated neurons in the ITV group was less than that of the IT group (10.3 ± 2.1 vs. 14.9 ± 2.3, P < 0.01), while the relative content level of POMC-derived peptides in the ITV group was half that of the IT group (P < 0.01). CONCLUSIONS: The celiac branches of the vagus might contribute to less eating and improvement of diabetes after IT. The activating vagus strategy might be a goal for the treatment of diabetes.

Changes of Ghrelin/GOAT axis and mTOR pathway in the hypothalamus after sleeve gastrectomy in obese type-2 diabetes rats.

AIM: To examine the changes of the ghrelin/ghrelin O-acyltransferase (GOAT) axis and the mammalian target of rapamycin (mTOR) pathway in the hypothalamus after sleeve gastrectomy. METHODS: A total of 30 obese type-2 diabetes Sprague-Dawley (SD) rats, 6 wk of age, fed with high-sugar and high-fat fodder for 2 mo plus intraperitoneal injection of streptozotocin were randomly divided into three groups: non-operation group (S0 group, n = 10), sham operation group (Sh group, n = 10) and sleeve gastrectomy group (SG group, n = 10). Data of body mass, food intake, oral glucose tolerance test (OGTT), acylated ghrelin (AG) and total ghrelin (TG) were collected and measured at the first day (when the rats were 6 wk old), preoperative day 3 and postoperative week 8. The mRNA expression of preproghrelin, GOAT and neuropeptide Y (NPY), and protein expression of ghrelin, GOAT, GHSR and the mTOR pathway (p-Akt, p-mTOR and p-S6) were measured in the hypothalamus. RESULTS: SG can significantly improve metabolic symptoms by reducing body mass and food intake. The obese rats showed lower serum TG levels and no change in AG, but the ratio of AG/TG was increased. When compared with the S0 and Sh groups, the SG group showed decreased TG (1482.03 ± 26.55, 1481.49 ± 23.30 and 1206.63 ± 52.02 ng/L, respectively, P < 0.05), but unchanged AG (153.06 ± 13.74, 155.37 ± 19.30 and 144.44 ± 16.689 ng/L, respectively, P > 0.05). As a result, the ratio of AG/TG further increased in the SG group (0.103 ± 0.009, 0.105 ± 0.013 and 0.12 ± 0.016, respectively, P < 0.05). When compared with the S0 group, SG suppressed mRNA and protein levels of preproghrelin (0.63 ± 0.12 vs 0.5 ± 0.11, P < 0.05) and GOAT (0.96 ± 0.09 vs 0.87 ± 0.08, P < 0.05), but did not change NPY mRNA expression (0.61 ± 0.04 vs 0.65 ± 0.07, P > 0.05) in the hypothalamus. The protein levels of p-Akt, p-mTOR and p-S6 were higher in the SG group, which indicated that the hypothalamic mTOR pathway was activated after SG at the postoperative week 8. CONCLUSION: The reduction of ghrelin expression and activation of the mTOR pathway might have opposite effects on food intake, as SG improves obesity and T2DM.

Red Ginseng Administration Before Islet Isolation Attenuates Apoptosis and Improves Islet Function and Transplant Outcome in a Syngeneic Mouse Marginal Islet Mass Model.

BACKGROUND: Transplantation of isolated islets is a promising treatment for diabetes. Red ginseng (RG) is steamed ginseng and has been reported to enhance insulin secretion-stimulating and anti-apoptotic activities in pancreatic ß-cells. In this study, we examined the hypothesis that pre-operative RG treatment enhances islet cell function and anti-apoptosis and investigated whether RG improves islet engraftment by transplant of a marginal mass of syngeneic islets pretreated with RG in diabetic mice. METHODS: Balb/c mice were randomly divided into 2 groups, and 1 group was administered RG (400 mg/kg/day orally) for 7 days before islet isolation. In vitro islet viability and function were assessed. After cytokine treatment, cell viability, function, and apoptosis of islet cells were analyzed. Furthermore, we studied the effects of RG in a syngeneic islet graft model. A marginal mass of syngeneic mouse islets was transplanted into diabetic hosts. RESULTS: Islet pretreatment with RG showed 1.4-fold higher glucose-induced insulin secretion than did control islets. RG pretreatment upregulated B-cell lymphoma 2 (Bcl-2) expression and downregulated Bcl-associated X protein (BAX), caspase-3, and inducible nitric oxide synthase (iNOS) expression. Glucose-induced insulin release, NO, and apoptosis were significantly improved in RG-pretreated islets compared with cytokine-treated islets. RG-pretreated mice exhibited improved marginal mass islet graft survival compared with controls. CONCLUSIONS: These results suggest that pre-operative RG administration enhanced islet function before transplantation and attenuated cytokine-induced damage associated with apoptosis. These studies indicate that inhibition of apoptosis by RG significantly improved islet cell and graft function after isolation and transplantation, respectively.

Characterisation of insulin-producing cells differentiated from tonsil derived mesenchymal stem cells.

Tonsil-derived (T-) mesenchymal stem cells (MSCs) display mutilineage differentiation potential and self-renewal capacity and have potential as a banking source. Diabetes mellitus is a prevalent disease in modern society, and the transplantation of pancreatic progenitor cells or various stem cell-derived insulin-secreting cells has been suggested as a novel therapy for diabetes. The potential of T-MSCs to trans-differentiate into pancreatic progenitor cells or insulin-secreting cells has not yet been investigated. We examined the potential of human T-MSCs to trans-differentiate into pancreatic islet cells using two different methods based on ß-mercaptoethanol and insulin-transferin-selenium, respectively. First, we compared the efficacy of the two methods for inducing differentiation into insulin-producing cells. We demonstrated that the insulin-transferin-selenium method is more efficient for inducing differentiation into insulin-secreting cells regardless of the source of the MSCs. Second, we compared the differentiation potential of two different MSC types: T-MSCs and adipose-derived MSCs (A-MSCs). T-MSCs had a differentiation capacity similar to that of A-MSCs and were capable of secreting insulin in response to glucose concentration. Islet-like clusters differentiated from T-MSCs had lower synaptotagmin-3, -5, -7, and -8 levels, and consequently lower secreted insulin levels than cells differentiated from A-MSCs. These results imply that T-MSCs can differentiate into functional pancreatic islet-like cells and could provide a novel, alternative cell therapy for diabetes mellitus.

Dietary zinc supplementation to the donor improves insulin secretion after islet transplantation in chemically induced diabetic rats.

OBJECTIVES: Zinc (Zn) is related to insulin synthesis, storage, and secretion. This study demonstrates the effects of Zn supplementation in donor rats on the outcomes of islet transplantation. METHODS: Donor rats received 3 different regimens of dietary Zn supplementation for 2 weeks before undergoing pancreas donation: a standard diet containing Zn at 50 ppm (control), 1 ppm (low-Zn group) or 1000 ppm (high-Zn group), respectively. Diabetic recipient rats underwent islet transplantation, and the blood glucose levels and insulin secretion were monitored for 7 days after transplantation. RESULTS: The serum and pancreatic Zn levels at the time of donation were significantly lower in the low-Zn group (48.8 ± 25.5 µg/dL and 11.3 ± 1.9 µg/g) and higher in the high-Zn group (147.3 ± 17.6 µg/dL and 18.7 ± 2.2 µg/g) when compared with those observed in the controls (118.7 ± 7.9 µg/dL and 14.6 ± 2.0 µg/g) (P < 0.05). The blood glucose levels became re-elevated 2 days after transplantation in rats receiving islet grafts from the controls and the low-Zn groups. In contrast, in the rats that received islets from the high-Zn groups, these were maintained within a reference range (P < 0.01). CONCLUSIONS: These data indicate that a Zn-rich diet for donor rats improves the function of islet grafts in chemically induced diabetic rats.

CXCR1/2 inhibition enhances pancreatic islet survival after transplantation.

Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1/2 axis in mice improved intrahepatic islet engraftment and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation.

Effects of astragalosides on cultured islets after cryopreservation in rats.

OBJECTIVE: To explore the effects of AST (astragalosides) on cultured rat islet yield, purity, and function after cryopreservation in rats. METHODS: Pancreatic islets were isolated from 30 Sprague-Dawley rats using the standard technique of collagenase P digestion and discontinuous Ficoll gradient purification. After thaw, the islets were randomly divided into AST group and control group (n=15). Next, the islet cells were cultured in AST-containing medium or standard medium for 7, 14, and 21 days after cryopreservation and thaw. The quantity, purity, and survival rate were calculated in the two groups before and after culture. Then the in vitro and in vivo function was observed in diabetic rats after islet transplantation. RESULTS: The quantity and purity of islets had no difference between the two groups before culture (P>.05) while the difference after culture was significantly (P<.05). The survival rate of islets was 48% in AST group and 32% in the control group 21 days after thaw (P<.05). After 3 days, there was significantly a higher simulation index in the AST group than in the control group (P<.05). There was a significant difference in blood glucose and insulin concentrations between the groups after 3 days (P<.05). CONCLUSION: AST can be added to the culture medium to reduce the loss of islet cryopreservation and be intravenously injected to improve culture islet function in vitro and prolong islet graft survival in diabetic rats.

Effect of zinc on early graft failure following intraportal islet transplantation in rat recipients.

BACKGROUND: Zinc (Zn) is related to insulin synthesis, storage and secretion. Zn status in patients with Type 1 diabetes is significantly lower than in healthy controls. Intraportal islet transplantation (IPIT) is a radical treatment for diabetes, the success of which depends largely on the survival of the transplanted islets. This study demonstrates the impact of a Zn-rich environment on transplanted islet survival. MATERIAL/METHODS: Diabetic Wistar rats were transplanted with syngeneic islets. Rats in the high-Zn-diet group were fed a standard pelleted diet containing ZnSO4 at 1000 ppm, whereas those in the control group were fed an ordinary diet alone (ZnSO4 at 50 ppm) for two weeks prior to islet transplantation. We examined Zn level of plasma, the blood glucose levels, and histological findings, etc., after intraportal islet transplantation. RESULTS: The high-Zn-diet group showed excellent blood glucose control compared with the control group on observation days 3, (237.1±120.6 mg/dl vs. 164.2±69.1 mg/dl; p<0.05) and 14, (273.7±160.9 mg/dl vs. 179.2±114.3 mg/dl; p<0.05). Early graft failure was found to be suppressed in the high-Zn-diet group on day 3 after transplantation (6.7% vs. 33.3%: p<0.05). As for the percentage of granulated islets, the high-Zn-diet group was improved (65.1% vs. 41.8%: p<0.05). CONCLUSIONS: These results indicated that a zinc-rich environment is advantageous for the recipient in intraportal islet transplantation. Zn is harmless in humans; thus, we consider that Zn supplementation could be a simple way to improve clinical results of intraportal islet transplantation.

Implantation of bFGF-treated islet progenitor cells ameliorates streptozotocin-induced diabetes in rats.

AIM: To examine whether implantation of islet preparation-derived proliferating islet cells (PIC) could ameliorate diabetes in rats. METHODS: PIC were expanded from rat islet preparation by supplementation of basic fibroblast growth factor (bFGF) and implanted into rats with streptozotocin (STZ)-induced diabetes through the portal vein. Body weight and blood glucose levels were measured. Serum insulin levels were measured by radioimmunoassay. The presence of insulin-positive cells was determined by hematoxylin and immunohistochemical staining. RESULTS: Cultured islet cells (CIC) were demonstrated to dedifferentiate in vitro, and the apoptosis ratios reached more than 50% by the 15th day post-isolation. PIC cells treated with bFGF (20 ng/mL) continued growing within 30 days after isolation, and no apoptotic cells were detected. Implantation of PIC into diabetic rats was capable of ameliorating diabetes, in terms of the restoration of euglycemia, weight gain, improved glucose response and elevated serum insulin levels for up to 130 days. Livers derived from PIC-implanted rats were examined for insulin expression and single insulin-positive cells. In addition, most islets of PIC-implanted STZ-induced diabetic rats were intact at 130 days post-transplantation and comparable to those of normal rats. CONCLUSION: Implantation of bFGF-treated proliferating islet cells is a promising cellular therapeutic approach for diabetes.

DHP-derivative and low oxygen tension effectively induces human adipose stromal cell reprogramming.

BACKGROUND AND METHODS: In this study, we utilized a combination of low oxygen tension and a novel anti-oxidant, 4-(3,4-dihydroxy-phenyl)-derivative (DHP-d) to directly induce adipose tissue stromal cells (ATSC) to de-differentiate into more primitive stem cells. De-differentiated ATSCs was overexpress stemness genes, Rex-1, Oct-4, Sox-2, and Nanog. Additionally, demethylation of the regulatory regions of Rex-1, stemnesses, and HIF1alpha and scavenging of reactive oxygen species were finally resulted in an improved stem cell behavior of de-differentiate ATSC (de-ATSC). Proliferation activity of ATSCs after dedifferentiation was induced by REX1, Oct4, and JAK/STAT3 directly or indirectly. De-ATSCs showed increased migration activity that mediated by P38/JUNK and ERK phosphorylation. Moreover, regenerative efficacy of de-ATSC engrafted spinal cord-injured rats and chemical-induced diabetes animals were significantly restored their functions. CONCLUSIONS/SIGNIFICANCE: Our stem cell remodeling system may provide a good model which would provide insight into the molecular mechanisms underlying ATSC proliferation and transdifferentiation. Also, these multipotent stem cells can be harvested may provide us with a valuable reservoir of primitive and autologous stem cells for use in a broad spectrum of regenerative cell-based disease therapy.

Transplantation of pancreatic islets from hypothalamic obese rats corrects hyperglycemia of diabetic rats.

Pancreatic islets isolated from adult obese rats, obtained by neonatal treatment with monosodium L-glutamate (MSG), oversecrete insulin stimulated by glucose concentration. Whereas adult MSG obese rats are hyperinsulinemic, their pancreatic islets still secrete insulin after high glucose demand. This is crucial so that the animals do not become hyperglycemic. Islets from MSG obese rats were implanted in diabetic donor rats so that the capacity of islets in regulating blood glucose concentration could be evaluated. Hyperglycemic (glucose 22 to 34 mmol/L) rats obtained with streptozotocin (STZ) treatment were used as recipients. Islet donors consisted of control adult and MSG obese rats. Only 600 islets were transplanted via the portal vein to diabetic rats. During 4 days after the transplant, fed blood glucose was monitored. After 12 hours of fasting the rats were killed; their blood samples were used to measure glucose and insulin concentration; retroperitoneal fat pads were isolated and weighed to estimate body fat. Transplanted islets from MSG obese rats decreased of fed glucose levels by 34% in diabetic rats (P < .05); however, glucose levels still remained twofold higher than those of intact controls (P < .05). Similar to MSG islets, islets grafts from control rats provoked the same effects in diabetic rats. High fasting blood glucose and low insulin levels of diabetic rats were corrected by islet grafts. Transplantations were able to recover 40% of fat in diabetic rats. The results demonstrated that islets from MSG obese rats may regulate blood glucose concentrations in diabetic rats, and suggesting that their function was not permanently altered by the onset of obesity.

The possible study of HuaXi-1 solution for all intra-abdominal organ preservation: a study in rats.

OBJECTIVE: University of Wisconsin (UW) solution continues to be the most commonly used solution for intra-abdominal organs. However, it is expensive. Therefore, we have formulated HuaXi-1 (HX-1) solution. The aim of this research was to assess the effect of HX-1 solution compared to UW, Collins 2, and hypertonic citrate solutions on intra-abdominal organs. METHODS: The effects of HX-1 solution for all intra-abdominal organ preservation were studied in a noncirculated, isolated, perfused rat liver model, in rat pancreas isografts in diabetic rats, and in a kidney autotransplant model. The effects were investigated by measuring hepatic tissue water content, sinusoidal lining cell mortality, Krebs-Henseleits perfusate, aspartate aminotransferase, the number of livers secreting bile during isolated perfusion, blood glucose, glucose tolerance tests, serum insulin of the rat pancreas-transplant recipients, the maximum serum creatinine levels, kidney graft survival rates, and observing the morphological changes in liver, pancreas, and kidneys. RESULTS: HX-1 solution preserved rat liver well for 24 hours as effectively as UW, rat pancreas as well for 48 hours, and dog kidney as well for 72 hours. CONCLUSION: The experimental findings indicated that HX-1 solution was effective to preserve all intra-abdominal organs; it may simplify cold storage of organs for transplantation.

Cutting edge: elimination of an endogenous adjuvant reduces the activation of CD8 T lymphocytes to transplanted cells and in an autoimmune diabetes model.

The generation of adaptive immune responses is thought to require the presence of adjuvants. Although microbial adjuvants are well characterized, little is known about what provides the adjuvant effect in responses to transplanted cells or in autoimmune diseases. It had been postulated that, in these situations, injured cells instead released "endogenous adjuvants." We previously identified uric acid as an endogenous adjuvant for coinjected Ags. We now report that elimination of uric acid reduced the generation of CTL to an Ag in transplanted syngeneic cells and the proliferation of autoreactive T cells in a transgenic diabetes model. In contrast, uric acid depletion did not reduce the stimulation of T cells to mature APCs or when endogenous APCs were activated with anti-CD40 Ab. These findings support the concept that danger signals contribute to the T cell responses to cell-associated Ags by activating APCs and identify uric acid as one of these signals.

Caspase-3 inhibitor prevents apoptosis of human islets immediately after isolation and improves islet graft function.

OBJECTIVES: Apoptosis appears in islets after isolation, and it has a detrimental effect on the islet function. To improve the outcome of clinical islet transplantation, it is crucial to protect islets from apoptosis. The aim of this study was to determine whether a caspase-3 inhibitor (Z-DEVD-FMK) added to culture media protects islets from apoptosis and to compare the effects of fetal bovine serum (FBS) with human serum albumin (HSA) as a protein supplement in culture. METHODS: Isolated human islets were cultured under 4 different conditions: 0.5% HSA (control), 0.5% HSA + 25 micromol/L Z-DEVD-FMK, 0.5% HSA + 100 micromol/L Z-DEVD-FMK and 10% FBS for 2 days. Next, 1000 IEQ islets precultured with 0.5% HSA and with or without 100 micromol/L Z-DEVD-FMK were transplanted to diabetic nude mice. RESULTS: The islet yields were higher in Z-DEVD-FMK-treated groups, and the inhibitor prevented apoptosis dose dependently. The yield and insulin release were higher in FBS-treated group than in the control group, but FBS did not affect apoptosis. All 6 mice transplanted with islets pretreated with Z-DEVD-FMK, and 3 of 8 mice with control islets became normoglycemic posttransplantation. CONCLUSION: Z-DEVD-FMK prevented apoptosis of isolated human islets and improved its function. FBS (10%) improved the islet yield and insulin secretion more than 0.5% HSA.

Beneficial effects of hyperbaric oxygen therapy on islet transplantation.

We envisage that hyperbaric oxygen (HBO) would ameliorate islet anoxia, preventing early graft failure. Thus, treatment of HBO to diabetic recipients should improve the outcome of islet transplantation. We tested this hypothesis by syngeneically transplanting insufficient number of islets (150 islets) into streptozotocin-diabetic C57BL/6 mice, each followed by HBO (2.4 ATA, 100% O2) therapy for 1.5 h from day 0 to 28, once daily (group A) or twice daily (group B), or from day 5 to 28, once daily (group C) or twice daily (group D), 6 days/week. Recipients without HBO treatment served as controls. At day 28 after transplantation, groups B, C, and D gained weight and had lower blood glucose compared with their baseline values. In addition, groups B and D had higher insulin content of the graft than the controls. To determine the optimal timing of HBO therapy, groups B and D were compared with recipients treated with HBO twice daily, 6 days/week, from day -14 to 0 (group E) and from day -14 to 28 (group F). At day 28 after transplantation, groups B, D, E, and F had significantly lower blood glucose than their individual baseline values and higher insulin content of the graft than controls. But only group F had more beta-cell mass of the graft than controls. These findings lend credence to the expectation that peritransplant application of adequate frequency of HBO to diabetic recipients would enhance the performance and growth of the islet graft, resulting in an improvement of the outcome of the transplantation.

Blockade of the CD28 and CD40 pathways result in the acceptance of pig and rat islet xenografts but not rat cardiac grafts in mice.

Previously, we demonstrated that combination CTLA4-Fc and anti-CD40L mAb treatment results in tolerance to concordant, cellular islet xenografts. The aim of this study was to determine its effectiveness in a model of fetal pig pancreas (FPP) xenotransplantation. Survival of FPP fragment grafts were compared to the survival of rat islet or cardiac xenografts following short term CTLA4-Fc and anti-CD40L mAb treatment. Rat islet and FPP fragment grafts survived long-term. However, rat cardiac grafts were rejected by 52-91 days. Both rat islet and FPP grafts showed similar histology with intact islet structures and adjacent 'nests' of lymphocytes. Concordant vascularised rat hearts showed extensive polymorphonuclear infiltrate, concentric vasculitis and a perivascular infiltrate predominantly of CD8+ T cells. This suggests that this therapy is effective for prolonging islet xenografts and demonstrates that the cellular mechanism of rejection for vascularised and non-vascularised xenografts are different.

An antagonist IL-15/Fc protein prevents costimulation blockade-resistant rejection.

IL-15 is a powerful T cell growth factor (TCGF) with particular importance for the maintenance of CD8(+) T cells. Because costimulation blockade does not result in universal tolerance, we hypothesized that "escape" from costimulation blockade might represent a CD8(+) and IL-15/IL-15R(+)-dependent process. For this analysis, we have used an IL-15 mutant/Fcgamma2a protein, a potentially cytolytic protein that is also a high-affinity receptor site specific antagonist for the IL-15Ralpha receptor protein, as a therapeutic agent. The IL-15-related fusion protein was used as monotherapy or in combination with CTLA4/Fc in murine islet allograft models. As monotherapies, CTLA4/Fc and an IL-15 mutant/Fcgamma2a were comparably effective in a semiallogeneic model system, and combined treatment with IL-15 mutant/Fcgamma2a plus CTLA4/Fc produced universal permanent engraftment. In a fully MHC-mismatched strain combination known to be refractory to costimulation blockade treatment, combined treatment with both fusion proteins proved to be highly effective; >70% of recipients were tolerized. The analysis revealed that the IL-15 mutant/Fc treatment confers partial protection from both CD4(+) and CD8(+) T cell graft infiltration. In rejections occurring despite CTLA4/Fc treatment, concomitant treatment with the IL-15 mutant/Fcgamma2a protein blocked a CD8(+) T cell-dominated rejection processes. This protection was linked to a blunted proliferative response of alloreactive T cells as well silencing of CTL-related gene expression events. Hence, we have demonstrated that targeting the IL-15/IL-15R pathway represents a new and potent strategy to prevent costimulation blockade-resistant CD8(+) T cell-driven rejection.

Syngeneic pancreatic islet transplantation reverses endothelial dysfunction in experimental diabetes.

Diabetes is known to cause impaired endothelium-dependent relaxation of blood vessels. The purpose of this study was to determine whether this endothelial dysfunction is a permanent defect or is reversible after acute arginine supplementation in vitro or by surgical intervention in vivo using syngeneic pancreatic islet transplantation. Lewis rats were injected with streptozotocin to induce diabetes and were studied either 8 or 12 weeks later. Another group received syngeneic islets via intraportal injection at 8 weeks of diabetes and were allowed to become euglycemic for 4 weeks before study. Thoracic aortic rings were tethered in isolated muscle baths, contracted with a submaximal concentration of norepinephrine, and challenged with either the endothelium-dependent vasodilator acetylcholine or the endothelium-independent vasodilator nitroglycerin. Relaxation to acetylcholine (but not nitroglycerin) was reduced in both 8- and 12-week diabetic rings compared with age-matched control rings. Preincubation of diabetic rings in vitro with L-arginine (but not D-arginine) restored relaxation to acetylcholine to normal to rings from 8-week but not 12-week diabetic animals. Plasma basic amino acids (arginine, lysine, and histidine) were reduced by diabetes, whereas other neutral or acidic amino acids were unchanged (phenylalanine, proline, and glutamate), reduced (serine, cysteine, threonine, tyrosine, tryptophan, and aspartate), or elevated (isoleucine, leucine, and valine). Islet transplantation restored to normal the changes in plasma amino acids. Elevation in blood glucose and total glycosylated hemoglobin in diabetic animals was normalized after islet transplantation. Furthermore, islet transplantation completely restored the defective endothelium-dependent relaxation to acetylcholine in diabetic rings.(ABSTRACT TRUNCATED AT 250 WORDS)