Active Subjects With Autoimmune Type 1 Diabetes Have Better Metabolic Profiles Than Sedentary Controls.

Previous studies in humans with type 1 diabetes mellitus (T1D) and in nonobese diabetic mice have investigated the beneficial immunomodulatory potential of aerobic physical activity. Performing high volume of aerobic exercise may favorably regulate autoimmunity in diabetes. We tested whether increased physical activity is a self-sufficient positive factor in T1D subjects. During a 3-month observational period, active (six males; 40.5 ± 6.1 years; BMI: 24.5 ± 2.1) and sedentary (four males, three females; 35.9 ± 8.9 years; BMI: 25.7 ± 3.8) T1D individuals on insulin pump therapy were studied for metabolic, inflammatory, and autoimmune parameters. At baseline and at the end of a 3-month period, glycosylated hemoglobin (HbA1c), autoantibodies (anti-GAD, anti-ZnT8, anti-IA2, and ICA) and proinflammatory cytokines (IL-6 and TNF-α) were evaluated. During the third month of the period, physically active T1D patients showed a significant reduction in the average glucose levels (-9%, p = 0.025, by CGM) compared to the first month values, and even their hyperglycemic episodes (>180 mg/dl) diminished significantly (-24.2%, p = 0.032 vs. first month). Moreover, active T1D subjects exhibited an improved body composition with respect to sedentary controls. No significant changes were detected as to the autoimmune and inflammatory profiles. This study confirms the beneficial role of physical exercise associated with insulin pump therapy in order to improve metabolic control in individuals with T1D. These preliminary positive observations need to be challenged in a prolonged interventional follow-up.

The anti-inflammatory effects of exercise in the syndromic thread of diabetes and autoimmunity.

A unifying thread over the wide spectrum of diabetes might be the triggering of innate immunological and inflammatory pathways leading to insulin resistance, beta-cell dysfunction and beta-cell destruction: the hybrid features of type 1 and type 2 diabetes. In fact, hyperglycemia can arise from a deficit in insulin action, insulin secretion, or both. Regularly exercising at moderate intensity has been shown to efficiently and positively impact upon physiological imbalances caused by several morbid conditions. Even in different immunological dysfunctions, physical exercise has been prescribed as a complementary therapeutic strategy. In fact, as suggested by our observations, there is a putative inverse relationship between autoimmunity markers (GAD, IA) and exercise-derived energy expenditure in type 1 pre-diabetic subjects. Exercise also has been shown to maintain muscle mitochondrial function and thus ability to maintain fuel metabolism and islet cell function. An additional benefit is the enhancement of antioxidant defense system and thus reducing oxidative stress. Therefore, the purpose of this review is to address the importance of physical exercise in a broad range of metabolic disorders that set out a common milieu in which type 1 and type 2 diabetes could be identified as one extensive syndrome.

Cellular Therapies and Regenerative Strategies for Diabetes - Proceeding of the STEMSO Conference.

Diabetes is now considered a growing global epidemic with sizable negative effects on patients' quality of life and life expectancy, and escalating economic impact (41% growth in the past five year), now representing health care ex-penditure impact of 256 Billion/year in the US alone. Objectives of cellular therapies and regenerative medicine strategies for treatment of diabetes are to reverse the disease condition and prevent the development of the severe chronic complications that can affect most organ systems in a large proportion of patients over time. Cell based therapies include the combination of immunomodulatory approaches aimed at restoring self tolerance (i.e., in the case of autoimmune diabetes) and at inducing permanent acceptance of transplanted tissues (in the case of allogeneic donors), or immune protection (i.e., engineered microenvironment and/or encapsulation) so that the immune system can no longer destroy the new insulin producing cells introduced either by regenerating, reprogramming or replacement. Several approaches are currently under evaluation for restoration of beta cell mass. The prototype strategy for Replacement is pancreatic islet transplantation, which is now an approved procedure in several countries. Reprogramming from non insulin-producing cells or Regeneration strategies could represent an appealing alternative to overcome shortage of deceased donor organs for transplantation. The selection of the most appropriate source for insulin producing cells is still not defined and the selected alternatives between replacement, reprogramming and regeneration strategies will be further developed in pre-clinical model systems and pilot clinical trials, while carefully assessing safety, efficacy and cost-effectiveness, as well as the challenges imposed by scaling up the selected technologies to meet the demand of the millions of affected patients who could benefit from these strategies.

Evaluation of viable ß-cell mass is useful for selecting collagenase for human islet isolation: comparison of collagenase NB1 and liberase HI.

The selection of enzyme blend is critical for the success of human islet isolations. Liberase HI collagenase (Roche) was introduced in the 1990s and had been widely used for clinical islet transplantation. More recently, a blend collagenase NB1 has been rendered available. The aim of this study was to evaluate the isolation outcomes and islet quality comparing human islet cells processed using NB1 and Liberase HI. A total of 90 isolations processed using NB1 (n = 40) or Liberase HI (n = 50) was retrospectively analyzed. Islet yield, function in vitro and in vivo, cellular (including ß-cell-specific) viability and content, as well as isolation-related factors were compared. No significant differences in donor-related factors were found between the groups. There were also no significant differences in islet yields (NB1 vs. Liberase: 263,389 ± 21,550 vs. 324,256 ± 27,192 IEQ; p = n.s., respectively). The pancreata processed with NB1 showed a significantly longer digestion time (18.6 ± 0.7 vs. 14.5 ± 0.5 min, p < 0.01), lower ß-cell viability (54.3 ± 3.4% vs. 72.0 ± 2.1%, p < 0.01), ß-cell mass (93,671 ± 11,150 vs. 148,961 ± 12,812 IEQ, p < 0.01), and viable ß-cell mass (47,317 ± 6,486 vs. 106,631 ± 10,228 VßIEQ, p < 0.01) than Liberase HI. In addition, islets obtained with Liberase showed significantly better graft function in in vivo assessment of islet potency. The utilization of collagenase NB1 in human islet isolation was associated with significantly lower ß-cell viability, mass, and islet potency in vivo in our series when compared to Liberase HI, even though there was no significant difference in islet yields between the groups. Evaluation of viable ß-cell mass contained in human islet preparations will be useful for selecting enzyme blends.

Immunomodulatory properties of porcine, bone marrow-derived multipotent mesenchymal stromal cells and comparison with their human counterpart.

Thanks to their immunonodulatory properties, multipotent mesenchymal stromal cells (MSCs) are a promising strategy for preventing/reducing the risk of graft rejection after hematopoietic cell and solid organ transplantation. We have previously demonstrated that porcine MSCs (pMSCs) can be isolated from bone marrow and display similar morphology and differentiative capacity as compared to human MSC (hMSCs). In this study, we investigated the in vitro immunomodulatory properties (namely the ability to suppress lymphocyte proliferation in response to phytohemagglutinin and the cytokine production in the culture supernatants) of pMSCs from six Large White 6-month old piglets. Similarly to hMSCs, pMSCs reduced the phytohemagglutinin-induced lymphocyte proliferation. High levels of IL-6 were found in culture supernatants, whereas IL-10 and TGF-ß were not detectable. In conclusion, ex vivo expanded pMSCs share selected biological/functional properties with hMSCs. pMSCs may be used in in vivo models to investigate novel approaches of prevention of graft rejection in solid organ transplantation.

In vivo induction of myeloid suppressor cells and CD4(+)Foxp3(+) T regulatory cells prolongs skin allograft survival in mice.

Natural CD4(+)Foxp3(+) T regulatory (Treg) cells can promote transplantation acceptance across major histocompatibility complex (MHC) barriers, while myeloid-derived suppressor cells (MDSCs) inhibit effector T-cell responses in tumor-bearing mice. One outstanding issue is whether combining the potent suppressive function of MDSCs with that of Treg cells might synergistically favor graft tolerance. In the present study, we evaluated the therapeutic potential of MDSCs and natural Treg cells in promoting allograft tolerance in mice by utilizing immunomodulatory agents to expand these cells in vivo. Upon administration of recombinant human granulocyte-colony stimulating factor (G-CSF; Neupogen), or interleukin-2 complex (IL-2C), Gr-1(+)CD11b(+) MDSCs or CD4(+)Foxp3(+) Treg cells were respectively induced at a high frequency in the peripheral lymphoid compartments of treated mice. Interestingly, induced MDSCs exhibited a more potent suppressive function in vitro when compared to MDSCs from naive mice. Furthermore, in vivo coadministration of Neupogen and IL-2C induced MDSCs at percentages that were higher than those seen when either agent was administered alone, suggesting an additive effect of the two drugs. Although treatment with either IL-2C or Neupogen led to a significant delay of MHC class II disparate allogeneic donor skin rejection, the combinatorial treatment was superior to either alone. Importantly, histological assessment of surviving grafts revealed intact morphology and minimal infiltrates at 60 days posttransplant. Collectively, our findings demonstrate that concurrent induction of MDSCs and Tregs is efficacious in downmodulating alloreactive T-cell responses in a synergistic manner and highlight the therapeutic potential of these naturally occurring suppressive leukocytes to promote transplantation tolerance.

Antiproinflammatory effects of iodixanol (OptiPrep)-based density gradient purification on human islet preparations.

Islet isolation and purification using a continuous density gradient may reduce the volume of tissue necessary for implantation into patients, therefore minimizing the risks associated with intraportal infusion in islet transplantation. On the other hand, the purification procedure might result in a decreased number of islets recovered due to various stresses such as exposure to cytokine/chemokine. While a Ficoll-based density gradient has been widely used in purification for clinical trials, purification with iodixanol (OptiPrep) has been recently reported in islet transplant series with successful clinical outcomes. The aim of the current study was to compare the effects of the purification method using OptiPrep-based and Ficoll-based density gradients. Human islet isolations were performed using a modified automated method. After the digestion phase, pre-purification digests were divided into two groups and purified using a semiautomated cell processor with either a continuous Ficoll- or OptiPrep-based density gradient. The quantity, purity, viability, and cellular composition of islet preparations from each group were assessed. Cytokine/chemokine and tissue factor production from islet preparations after 48-h culture were also measured. Although islet purity, post-purification IEQ, islet recovery rate, FDA/PI, and fractional ß-cell viability were comparable, ß-cell mass after 48-h culture significantly improved in the OptiPrep group when compared to the Ficoll group. The production of cytokine/chemokine including IL-1ß, TNF-α, IFN-γ, IL-6, IL-8, MIP-1ß, MCP-1, and RANTES but not tissue factor from the OptiPrep group was significantly lower during 48-h culture after isolation. Each preparation contained the similar number of ductal cells and macrophages. Endotoxin level in both gradient medium was also comparable. The purification method using OptiPrep gradient media significantly reduced cytokine/chemokine production but not tissue factor from human islet preparations and improved ß-cell survival during pretransplant culture. Our results suggest that the purification method using OptiPrep gradient media may be of assistance in increasing successful islet transplantation.

Feasibility of localized immunosuppression: 1. Exploratory studies with glucocorticoids in a biohybrid device designed for cell transplantation.

Emerging biotechnologies, such as the use of biohybrid devices for cellular therapies, are showing increasing therapeutic promise for the treatment of various diseases, including type 1 diabetes mellitus. The functionality of such devices could be greatly enhanced if successful localized immunosuppression regimens could be established, since they would eliminate the many otherwise unavoidable side effects of currently used systemic immunosuppressive therapies. The existence of local immune privilege at some specialized tissues, such as the eye, CNS, or pregnant uterus, supports the feasibility of localized immunomodulation, and such an approach is particularly well-suited for cell transplant therapies where all transplanted tissue is localized within a device. Following the success of syngeneic transplantation in a subcutaneous prevascularized device as a bioartificial pancreas in a rodent model, we now report the first results of exploratory in vivo islet allograft studies in rats using locally delivered glucocorticoids (dexamethasone phosphate and the soft steroid loteprednol etabonate). Following in vitro assessments, in silico drug distribution models were used to establish tentative therapeutic dose ranges. Sustained local delivery was achieved via implantable osmotic mini-pumps through a central sprinkler, as well as with a sustained-delivery formulation for loteprednol etabonate using poly(D,L-lactic) acid (PLA) microspheres. Doses delivered locally were approximately hundred-fold smaller than those typically used in systemic treatments. While several solubility, stability, and implantation problems still remain to be addressed, both compounds showed promise in their ability to prolong graft survival after tapering of systemic immunosuppression, compared to control groups.

Effects of systemic immunosuppression on islet engraftment and function into a subcutaneous biocompatible device.

The aim of this study was to explore the effect of sirolimus (Sir) and tacrolimus (Tac) on islets implanted into a subcutaneous (SC), prevascularized device in syngeneic rats. Animals received a 40-day treatment with Tac and Sir (alone or in combination) starting either on day 0 or 40 days after islet transplantation. Controls received no treatment. A 40-day washout period was performed after immunosuppression (IS). Glycemia and intravenous glucose tolerance tests (IVGTT) were assessed at follow-up. In the control group, 75% of recipients achieved stable normoglycemia after islet transplantation, while none reversed diabetes with any IS regimen started on day 0. Graft dysfunction was irreversible after IS withdrawal. Glucose clearance (IVGTT) was significantly impaired among Tac-treated compared with control groups (P < .05 with IS; P < .01 after washout). Among animals with established grafts, islet dysfunction which occurred under IS treatment persisted after washout in animals treated with Tac and Sir plus Tac. When compared with controls, glucose clearance was significantly impaired in the Tac and Tac plus Sir groups before and after IS (P < .01, Tac; P < 0.01, Tac plus Sir). Sir and Tac showed profound deleterious effects on islet cell engraftment and function, which may hinder the success of implantation into biohybrid devices. Nondiabetogenic IS protocols must be developed for clinical application of islet transplantation into biohybrid devices.

c-Jun N-terminal kinase 1 is deleterious to the function and survival of murine pancreatic islets.

AIMS/HYPOTHESIS: Inhibition of c-jun N-terminal kinase (JNK) favours pancreatic islet function and survival. Since two JNK isoforms are present in the pancreas (JNK1 and JNK2), we addressed their specific roles in experimental islet transplantation. METHODS: C57BL/6J (wild-type [WT]), Jnk1 (also known as Mapk8)(-/-) and Jnk2 (also known as Mapk9)(-/-) mice were used as donor/recipients in a syngeneic islet transplantation model. Islet cell composition, function, viability, production of cytokines and of vascular endothelial growth factor (VEGF) were also studied in vitro. RESULTS: Jnk1 ( -/- ) islets secreted more insulin in response to glucose and were more resistant to cytokine-induced cell death compared with WT and Jnk2 (-/-) islets (p < 0.01). Cytokines reduced VEGF production in WT and Jnk2 (-/-) but not Jnk1 ( -/- ) islets; VEGF blockade restored Jnk1 ( -/- ) islet susceptibility to cytokine-induced cell death. Transplantation of Jnk1 ( -/- ) or WT islets into WT recipients made diabetic had similar outcomes. However, Jnk1 ( -/- ) recipients of WT islets had shorter time to diabetes reversal (17 vs 55 days in WT, p = 0.033), while none of the Jnk2 (-/-) recipients had diabetes reversal (0% vs 71% in WT, p = 0.0003). Co-culture of WT islets with macrophages from each strain revealed a discordant cytokine production. CONCLUSIONS/INTERPRETATION: We have shown a deleterious effect of JNK2 deficiency on islet graft outcome, most likely related to JNK1 activation, suggesting that specific JNK1 blockade may be superior to general JNK inhibition, particularly when administered to transplant recipients.

Human alpha 1-antitrypsin therapy induces fatal anaphylaxis in non-obese diabetic mice.

Previous studies have shown that human alpha-1 antitrypsin (hAAT) gene delivery prevents type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. Furthermore, hAAT protein administration prolongs acceptance of islet allografts. Therefore, we evaluated the use of purified hAAT protein therapy to prevent T1D in NOD mice. Female NOD, non-obese resistant (NOR), Balb/c and C57BL/6 mice were injected intraperitoneally with vehicle alone or vehicle containing hAAT, human albumin or mouse albumin (or mg/injection/mouse; 2x/week). Preparations of clinical-grade hAAT included API(R), Aralast, Prolastin and Zemaira. Surprisingly, hAAT administration was associated with a high rate of fatal anaphylaxis. In studies seeking T1D prevention at 4 weeks of age, 100% mice died after six injections of hAAT. When administrated at 8-10 weeks of age, most (80-100%) NOD mice died following the fourth injection of hAAT, while 0% of Balb/c and C57BL/6 mice and 10% of NOR mice died. Interestingly, repeated injections of human albumin, but not mouse albumin, also induced sudden death in NOD mice. Antibodies to hAAT were induced 2-3 weeks after hAAT administration and death was prevented by treatment with anti-platelet-activating factor along with anti-histamine. In studies of disease reversal in NOD mice, using the four pharmaceutical grade formulations of hAAT, anaphylactic deaths were observed with all hAAT preparations. The propensity for fatal anaphylaxis following antigenic administration appears to be NOD- but not hAAT-specific. The susceptibility of NOD mice to hypersensitivity provides a significant limitation for testing of hAAT. Development of strategies to avoid this unwanted response is required to use this promising therapeutic agent for T1D.

Combined islet and hematopoietic stem cell allotransplantation: a clinical pilot trial to induce chimerism and graft tolerance.

To prevent graft rejection and avoid immunosuppression-related side-effects, we attempted to induce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem cell (HSC) infusion. Six patients with brittle type 1 Diabetes Mellitus received a single-donor allogeneic islet transplant (8611 +/- 2113 IEQ/kg) followed by high doses of donor HSC (4.3 +/- 1.9 x 10(6) HSC/kg), at days 5 and 11 posttransplant, without ablative conditioning. An 'Edmonton-like' immunosuppression was administered, with a single dose of anti-TNFalpha antibody (Infliximab) added to induction. Immunosuppression was weaned per protocol starting 12 months posttransplant. After transplantation, glucose control significantly improved, with 3 recipients achieving insulin-independence for a short time (24 +/- 23 days). No severe hypoglycemia or protocol-related adverse events occurred. Graft function was maximal at 3 months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough levels, whereas 4 completed 1-year follow-up with functioning grafts. Graft failure occurred within 4 months from weaning (478 +/- 25 days posttransplant). Peripheral chimerism, as donor leukocytes, was maximal at 1-month (5.92 +/- 0.48%), highly reduced at 1-year (0.20 +/- 0.08%), and was undetectable at graft failure. CD25+T-lymphocytes significantly decreased at 3 months, but partially recovered thereafter. Combined islet and HSC allotransplantation using an 'Edmonton-like' immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance.

Factors that affect human islet isolation.

More than 10,000 IEQ/kg recipient weight of islets is often necessary to achieve insulin independence in patients with type 1 diabetes mellitus. Several studies have identified high donor body mass index (BMI) and pancreas size as important factors for the success of human islet isolation. However, the donor shortage underscores the need to improve isolation outcomes from lower BMI pancreas donors and/or small pancreata. The aim of this study was to identify the critical factors that affect isolation outcome. We analyzed the data from 207 isolations performed from 2002 to 2006 with respect to donor characteristics, pancreas condition, and processing variables. More than 3000 IEQ/g pancreas weight was considered to be an acceptable isolation outcome. This goal was obtained from donors with a BMI >30 kg/m2 (P = .002). The pancreatic surface integrity was also a significant factor (P = .02). Moreover, longer digestion times (P = .04) and a greater proportion of trapped islets negatively affected success rates (P = .004). As previously reported, pancreata from high BMI donors were suitable for islet isolation and transplantation, as they yielded higher total islet particle numbers and higher IEQ/g. Although BMI and pancreas size are not controllable due to the organ donor shortage, factors such as pancreatic surface integrity, shorter digestion time, and lower proportions of trapped islets were found to be significant to obtain higher success rates. The development of better protocols and systematic training of processing/procurement teams will be of assistance to increase the number of successful human islet isolations.

beta-Cell specific cytoprotection by prolactin on human islets.

INTRODUCTION: Many cytoprotective agents have been reported to improve islet isolation and transplantation outcomes. However, several of these agents improve all cell subsets within an islet preparation; selection of non-beta-cell components (eg, acinar cells) may have a negative effect on beta-cell function and survival. In this study, we examined the effect of prolactin (PRL) supplementation in the culture medium to determine whether it exerted beta-cell-selective cytoprotection on islet viability and function. MATERIALS AND METHODS: Human islets were precultured with or without recombinant human PRL (500 microg/L) for 48 hours. The fractional viability and cellular composition of non-beta-cell and beta-cell-specific components were assessed using FACS and Laser Scanning Cytometry (LSC). Islet potency was assessed in vivo by transplantation into chemically induced diabetic immunodeficient mice. RESULTS: The relative viable beta-cell mass and the relative islet beta-cell content in the PRL group were 28% higher (P = .018) and 19% higher (P = .029) than the control group, respectively. All transplanted mice achieved normoglycemia in both groups, indicating that PRL treatment did not alter islet function. CONCLUSION: PRL treatment improved beta-cell-specific viability and survival of human islets in vitro. The development of novel beta-cell-specific cytoprotective strategies may be of assistance in improving islet transplantation.

Rapamycin impairs beta-cell proliferation in vivo.

During pregnancy a high rate of beta-cell proliferation occurs, making of this a useful model for the study of islet cell expansion in vivo. We used the murine pregnancy model to assess the effect of Rapamycin treatment on islet cell proliferation in vivo. Rapamycin is routinely used for the prevention of graft rejection in transplanted patients, including islet transplant recipients. As expected, pregnancy led to increased beta-cell proliferation, islet yield and skewing in size distribution after isolation and pancreatic insulin content, when compared to non-pregnant females. Rapamycin treatment resulted in reduced beta cell proliferation in pregnant mice, while minimal effects of Rapamycin treatment were observed on islet function both in vivo and in vitro. Rapamycin treatment of islets resulted in reduced phosphorylation of p70s6k, a downstream effector molecule of mTOR and increased ERK1/2 phosphorylation. In conclusion, beta-cell replication is reduced under Rapamycin treatment in vivo, suggesting that this mechanism may be operational and impair beta-cell renewal in transplanted patients.

Prolonged islet allograft survival by alpha-1 antitrypsin: the role of humoral immunity.

Immunomodulatory properties have been recognized for human alpha-1 antitrypsin (hAAT). However, production of anti-hAAT antibodies in mice may inactivate the protein. In this study, we evaluated the effects of chronic hAAT administration on allogeneic islet graft survival. Chemically diabetic mice lacking an efficient humoral response due to the targeted disruption of the Ig mu-chain (muMT mice) or wild-type (WT) C57BL/6 mice received DBA/2 mouse islets under the kidney capsule. hAAT (Prolastin or Aralast) was given intraperitoneally on day 0 and every 3 days thereafter. Control animals received no treatment. hAAT administration in WT mice resulted in prolongation of islet allograft survival in a dose-dependent fashion in both hAAT-treated groups. Lack of Ig response (muMT mice) per se conferred a beneficial effect on graft survival that worsened in the Prolastin-treated groups but improved in the Aralast-treated group. Our data indicate that systemic administration of hAAT results in prolongation of islet allograft survival. Absence of mature B cells and Ig mu-chain resulted in improved graft survival, pointing to a role for B cells in the rejection process in this model. Treatment with Prolastin worsened graft survival in muMT mice, whereas Aralast did improve it, suggesting a different efficacy and possible actions of the two drug formulations.

Alpha-1 antitrypsin treatment of spontaneously diabetic nonobese diabetic mice receiving islet allografts.

Alpha-1 antitrypsin (AAT) is a serine protease inhibitor able to prevent diabetes onset in nonobese diabetic (NOD) mice and to prolong islet allograft survival in a nonautoimmune murine model. In this study, we explored the effect of chronic administration of human AAT (hAAT) on allogeneic (C57BL/6) islet graft survival in spontaneously diabetic female NOD mice. Mice received intraperitoneal treatment with saline, Prolastin (1 or 2 mg/mouse) or Aralast (2 mg/mouse) on days -1, 0, 3, 6, and 9. Saline-treated mice rejected the grafts 10.0 +/- 2.5 days after transplantation (n = 9). Prolastin 1 mg (n = 9) and 2 mg (n = 3) resulted in rejection on 8.7 +/- 1.4 (not significant) and 13.0 +/- 4.3 days (P < .03), respectively. Aralast-treated mice showed prolongation of graft survival (13 +/- 5.9 days; n = 5; P < .03). Notably, repeated administrations of either hAAT formulation led to sudden death of a proportion of treated animals. Collectively, our preliminary data indicate that prolongation of islet allograft survival in the stringent autoimmune diabetic NOD mouse model can be achieved with hAAT monotherapy. The death of a proportion of treated animals may be consequent to immunization to hAAT and lethal hypersensitivity. Interestingly, this phenomenon was not observed in a non-autoimmune mouse strain (C57BL/6) despite extended hAAT treatment (>100 days).

Inhibition of c-jun N terminal kinase (JNK) improves functional beta cell mass in human islets and leads to AKT and glycogen synthase kinase-3 (GSK-3) phosphorylation.

AIMS/HYPOTHESIS: Activation of c-jun N-terminal kinase (JNK) has been described in islet isolation and engraftment, making JNK a key target in islet transplantation. The objective of this study was to investigate if JNK inhibition with a cell-permeable TAT peptide inhibitor (L-JNKI) protects functional beta cell mass in human islets and affects AKT and its substrates in islet cells. METHODS: The effect of L-JNKI (10 micromol/l) on islet count, mitochondrial membrane potential, glucose-stimulated insulin release and phosphorylation of both AKT and its substrates, as well as on reversal of diabetes in immunodeficient diabetic Nu/Nu mice was studied. RESULTS: In vitro, L-JNKI reduced the islet loss in culture and protected from cell death caused by acute cytokine exposure. In vivo, treatment of freshly isolated human islets and diabetic Nu/Nu mice recipients of such islets resulted in improved functional beta cell mass. We showed that L-JNKI activates AKT and downregulates glycogen synthase kinase-3 beta (GSK-3B) in human islets exposed to cytokines, while other AKT substrates were unaffected, suggesting that a specific AKT/GSK-3B regulation by L-JNKI may represent one of its mechanisms of cytoprotection. CONCLUSIONS/INTERPRETATION: In conclusion, we have demonstrated that targeting JNK in human pancreatic islets results in improved functional beta cell mass and in the regulation of AKT/GSK3B activity.

Shipment of human islets for transplantation.

The use of regional human islet cell processing centers (ICPC) supporting distant clinical islet transplantation programs (CITP) has proven successful in recent clinical trials. Standardization of islet shipping protocols is needed to preserve cell product identity, quantity, quality and sterility, and to meet criteria for transplantation. We evaluated the use of gas-permeable bags for human islet preparation shipment from a single ICPC to two remote CITPs. Product release tests (counts, purity, viability, sterility and potency) were performed at both centers using identical protocols to determine adequacy for transplantation.Thirty-five islet preparations were shipped either immediately after isolation (n = 20) or following culture (n = 15). Islet recovery rate after shipment was higher in cultured preparations, when compared to those not cultured (91.2 +/- 4.9% vs. 72.9 +/- 4.7%, respectively; p < 0.05), though the overall recovery rate based on isolation and pre-transplant counts was comparable (72.9 +/- 4.7% vs. 70.4 +/- 3.5%, respectively; p = N.S.). All preparations met product release criteria for transplantation. Additional experiments showed that gas-permeable bags led to improved recovery and potency, when compared to 50-mL conical tubes or to non-gas-permeable bags for shipment.Collectively, our data demonstrate that the use of gas-permeable bags is efficient for clinical-grade and should be preferred also for the shipment of research-grade islet preparations.