Jeffrey A. SoRelle

Withaferin A inhibits pro-inflammatory cytokine-induced damage to islets in culture and following transplantation

Aims/hypothesisBeta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type 1 diabetes and loss of transplanted islets. The nuclear factor κB (NF-κB) signalling pathway is a key regulator of beta cell stress response, survival and apoptosis. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, has been demonstrated to be a potent, safe, anti-inflammatory molecule that can inhibit NF-κB signalling. Therefore, we evaluated the ability of WA to protect mouse and human islets from the damaging effects of pro-inflammatory cytokines in vitro and following intraportal transplantation.MethodsMouse and human islets were treated with a cytokine cocktail, and NF-κB activation was measured by immunoblots, p65 nuclear translocation and chromatin immunoprecipitation of p65-bound DNA. Intraportal transplantation of a marginal mass of syngeneic mouse islets was performed to evaluate the in vivo protective effect of WA.ResultsTreatment with WA substantially improved islet engraftment of syngeneic islets (83% for infusion with 200 islets + WA; 0% for 200 islets + vehicle) in a mouse model of diabetes, compared with marginal graft controls with superior islet function in WA-treated mice confirmed by glucose tolerance test. Treatment of human and mouse islets with WA prevented cytokine-induced cell death, inhibited inflammatory cytokine secretion and protected islet potency.ConclusionsWA was shown to be a strong inhibitor of the inflammatory response in islets, protecting against cytokine-induced cell damage while improving survival of transplanted islets. These results suggest that WA could be incorporated as an adjunctive treatment to improve islet transplant outcome.

Correlation of Released HMGB1 Levels with the Degree of Islet Damage in Mice and Humans and with the Outcomes of Islet Transplantation in Mice

Establishing reliable islet potency assay is a critical and unmet issue for clinical islet transplantation. Recently, we reported that islets contained high levels of high mobility group box 1 (HMGB1) and damaged islets released HMGB1 in a mouse model. In this study, we hypothesized that the amount of released HMGB1 could reflect the degree of islet damage, and could predict the outcome of islet transplantation. Four groups of damaged mouse islets and three groups of damaged human islets were generated by hypoxic conditions. These islets were assessed by in vivo (transplantation) and in vitro (released HMGB1 levels, released C-peptide levels, PI staining, TUNEL staining, ATP/DNA, and glucose-stimulated insulin release test) assays. In addition, the ability of each assay to distinguish between noncured (n = 13) and cured (n = 7) mice was assessed. The curative rates of STZ-diabetic mice after receiving control, hypoxia-3h, hypoxia-6h, and hypoxia-24h mouse islets were 100%, 40%, 0%, and 0%, respectively. Only amounts of released HMGB1 and ratio of PI staining significant increased according to the degree of damages in both human and mouse islets. In terms of predictability of curing diabetic mice, amounts of released HMGB1 showed the best sensitivity (100%), specificity (100%), positive (100%), and negative predictive values (100%) among all the assays. The amount of released HMGB1 reflected the degree of islet damage and correlated with the outcome of islet transplantation in mice. Hence, released HMGB1 levels from islets should be a useful marker to evaluate the potency of isolated islets.

Safety and tolerability of the T-cell depletion protocol coupled with anakinra and etanercept for clinical islet cell transplantation

Islet cell transplantation (ICT) is a promising approach to cure patients with type 1 diabetes. We have implemented a new immunosuppression protocol with antithymoglobulin plus anti‐inflammatory agents of anakinra and eternacept for induction and tacrolimus plus mycophenolate mofetil for maintenance [T‐cell depletion with anti‐inflammatory (TCD‐AI) protocol], resulting in successful single‐donor ICT.

Alleviation of instant blood-mediated inflammatory reaction in autologous conditions through treatment of human islets with NF-κB inhibitors.

Background The instant blood-mediated inflammatory response (IBMIR) has been shown as a major factor that causes damage to transplanted islets. Withaferin A (WA), an inhibitor of nuclear factor (NF) &kgr;B, was shown to suppress the inflammatory response in islets and improve syngeneic islet graft survival in mice. We investigated how treating islets with NF-&kgr;B inhibitors affected IBMIR using an in vitro human autologous blood islet model. Methods Human islets were pretreated with or without NF-&kgr;B inhibitors WA or CAY10512 before mixing autologous blood in a miniaturized in vitro tube model. Plasma samples were collected at multiple time points and used for the measurement of C-peptide, proinsulin, thrombin-antithrombin (TAT) complex, and a panel of proinflammatory cytokines. Infiltration of neutrophils into islets was analyzed using immunohistochemistry. Results Rapid release of C-peptide and proinsulin was observed 3 hr after mixing islets and blood in the control group, but not in the NF-&kgr;B inhibitor–treated groups, whereas TAT levels were elevated in all three groups with a peak at 6 hr. Significant elevation of proinflammatory cytokines was observed in the control group after 3 hr, but not in the treatment groups. Significant inhibition of neutrophil infiltration was also observed in the WA group compared with the control (P<0.001) and CAY10512 (P<0.001) groups. Conclusions A miniaturized in vitro tube model can be useful in investigating IBMIR. The presence of NF-&kgr;B inhibitor could alleviate IBMIR, thus improving the survival of transplanted islets. Protection of islets in the peritransplant phase may improve long-term graft outcomes.

High-Mobility Group Box 1 Expressions in Hypoxia-Induced Damaged Mouse Islets

INTRODUCTION Discovering a new, accurate, and useful damage marker for isolated islets is critical for avoiding the transplantation of nontherapeutic preparations. Recently, we have reported that islets that contained uniquely high levels of high-mobility group box 1 (HMGB1) protein and cytokine induced damaged islets released HMGB1 in a mouse model. Islets are frequently exposed to hypoxic conditions during organ procurement, organ transportation, islet isolation, and islet storage before transplantation. In the present study, we analyzed HMGB1 expressions in hypoxia-induced damaged mouse islets. METHODS Damaged mouse islets were generated by hypoxic conditions (1% O2, 5% CO(2), and 94% N(2)). HMGB1 expressions and production levels were assessed by quantitative real-time polymerase chain reaction (PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA) studies. In vivo islet function was analyzed using transplantation assay using streptozotocin-induced diabetic mice. RESULTS HMGB1 was mainly stained in the nucleus in the intact islets; however, HMGB1 was present in not only the nucleus, but also the cytoplasm in hypoxia-induced damaged islets. HMGB1 messenger RNA (mRNA) levels were up-regulated in the hypoxia-induced damaged islets, suggesting that HMGB1 was intentionally generated during hypoxia. HMGB1 protein levels in the islets were gradually decreased with time under hypoxic conditions. The amount of released HMGB1 levels and the amount of released HMGB1 levels per hour were significantly increased in damaged (noncurable) islets. CONCLUSIONS When islets were damaged by hypoxic condition, HMGB1 was synthesized and released from hypoxia-induced damaged islets. The amount of released HMGB1 and/or the amount of released HMGB1 per hour might be a useful marker for detecting damaged islets and might be used for islet potency assay.

An Effective Method to Release Human Islets From Surrounding Acinar Cells With Agitation in High Osmolality Solution

INTRODUCTION Islet purification is mainly performed by the density gradient method. However, purification of the embedded islets that are surrounded by exocrine tissue should be difficult, because their density is similar to exocrine tissue. In this study, we performed chart review to assess the relationship between the ratio of embedded islets and efficacy of purification. Then, we tested several conditions of a new method to free the islets from surrounded exocrine tissues using high osmolality solution with gentle agitation. MATERIALS AND METHODS First, we performed chart review of our human islet isolation. Second, embedded islet-enriched human islet fractions (embedded islets >50%) were suspended in University of Wisconsin (UW) solution (UW group, 320 mOsm/kg/H(2)0) or osmolality-adjusted UW solution (400, 500, and 600 mOsm/kg/H(2)0; 400 group, 500 group, and 600 group, respectively). Each tube was gently shaken at 4°C. The tissue samples were taken before shaking and after 15, 30, and 60 minutes. Islet yield, percentage of embedded islets, and viabilities were assessed. RESULTS The chart review revealed that high ratio of embedded islets deteriorated the efficacy of islet purification. The islet yield in all groups except for the 600 group did not change at 15 minutes, but it decreased in all groups at 60 minutes. The average percentage of embedded islets before shaking was 62.6%. Although percentage of embedded islets were decreasing in all groups, it was < 20% at 15 minutes in the 500 and 600 groups whereas it was >44% in the UW group, which indicated that higher osmolality would have a greater effect. Viability was >95% in all groups at 30 minutes. CONCLUSIONS The embedded islets deteriorated the efficacy of islet purification. Gentle agitation of embedded islets in high osmolality (500 mOsm/kg/H(2)O, 15 minutes) could release islets from surrounded exocrine tissue.

Comparison of surface modification chemistries in mouse, porcine, and human islets.

Beta cell replacement therapy, the transplantation of isolated pancreatic islets by intraportal infusion, offers patients with brittle type 1 diabetes blood glucose regulation with a minimally invasive technique. Chemical modification of islets prior to transplantation, providing a nanothin barrier that potentially includes active protective compounds, has been proposed as a strategy to minimize the inflammatory and immune reactions that often significantly limit graft function and duration. Chemical modification also has the potential to allow the use of alternative sources of islets, such as porcine islets, for transplantation. This investigation compared three orthogonal covalent islet modification techniques across three species (human, porcine, and murine), using multiple measures to determine biocompatibility and effectiveness. All three conjugation chemistries were well tolerated, and the overall efficiency, gross uniformity, and stability of the surface modifications were dependent upon the conjugation chemistry as well as the islet source (human, porcine, or murine). Notably, the reductive modification of surface disulfides was shown to afford intense and long-lasting modification of human islets. This study demonstrates that murine, human, and porcine islets tolerate a variety of covalent modifications, that these modifications are relatively stable, and that the murine islet model may not be predictive for some chemical contexts.

Usefulness of the Secretory Unit of Islet Transplant Objects (SUITO) Index for Evaluation of Clinical Autologous Islet Transplantation

BACKGROUND Assessing the engrafted islet mass is important in evaluating the efficacy of islet transplantation. We previously demonstrated that the average secretory unit of islet transplant objects (SUITO) index within 1 month of allogeneic islet transplantation was an excellent predictor of insulin independence. However, the usefulness of the SUITO index for evaluating autologous islet transplantation has not been explored. The purpose of the present study was to assess the relationship between the SUITO index and clinical outcomes after total pancreatectomy followed by autologous islet transplantation. METHODS We performed 27 total pancreatectomies followed by autologous islet transplantation from October 2006 to January 2011. Cases were divided into an insulin-independent group (IIG; n = 12) and an insulin-dependent group (lDG; n = 15). The SUITO index was calculated by the formula [fasting C-peptide (ng/mL)/fasting glucose (mg/dL) -63] × 1,500. The average SUITO index within the first month of transplantation except for days 0, 1, and 2, maximum SUITO index, and most recent SUITO index were calculated in each case, and values were compared between the IIG and the IDG. RESULTS The average SUITO index within 1 month was significantly higher in the IIG than in the IDG (24.6 ± 3.4 vs 14.9 ± 2.0, respectively; P < .02). The maximum SUITO indices were 45.7 ± 7.7 in the IIG and 30.1 ± 8.1 in the IDG (not significant), and the recent SUITO indices were 36.9 ± 6.7 in the IIG and 22.8 ± 6.1 in the IDG (not significant). CONCLUSIONS The average SUITO index within 1 month was an excellent predictor of insulin independence after total pancreatectomy followed by autologous islet transplantation.

Beta Cell Replacement Therapy

Type 1 Diabetes mellitus (T1D) is an autoimmune disorder caused by destruction of pancreatic beta cells which produce insulin. Current estimates have shown that T1D affects about 0.4 to 0.8% of the population worldwide accounting for 5-10% of all diabetes cases (Stock and Bluestone, 2004). It is seen as an increasing health hazard. For T1D, exogenous insulin administration is the most widely used treatment. However, this treatment has several limitations including development of secondary health complications over time. There are several approaches already in practice to replace the damaged beta cells in T1D. They include whole organ pancreas or isolated islet cell transplantation. Several novel approaches are currently in development such as expansion of adult beta cells and stem/progenitor cells which can be transformed into beta cells. This review summarizes recent progress made in beta cell replacement therapy for T1D. Diabetes mellitus has been known for thousands of years. Diabetes comes from an ancient Greek word coined by Arataeus of Cappadocia, and mellitus from the Latin word honey associated with sweet urine. (Medvei, 1993). The pathogenesis of T1D has been attributed to an autoimmune response. Currently the mechanism that triggers this disease is still unknown, but it seems to be a combination of environmental and genetic factors. T-cells in the host’s body become sensitized to protein that naturally occurs in the beta cells of the pancreas and the immune system begins to mount a specific attack leading to destruction of the islets of Langerhans. Several candidate proteins have been identified including GAD65, insulin, and ZnT8 (Harlan et al., 2009, Shapiro et al., 2003). Reducing the autoimmune effect in beta cell replacement is very important, but before moving onto the strategies for beta cell replacement therapy it is important to describe the standard therapy for T1D and how it falls short of providing for all the needs of diabetic patients. Exogenous insulin therapy continues to be the leading therapy for T1D today, but there are several complications associated with it. However, even intensive monitoring of blood glucose and injection of insulin is not enough to halt the secondary complications of diabetes (Harlan et al., 2009). Diabetes mellitus can decrease patients’ lifespan, especially in severe cases. Lowered blood sugar levels, which can result if too much insulin is administered, can also lead to a hypoglycemic episode (Cooke and Plotnick, 2008). These episodes are typified by ketoacidosis, where in an effort to find energy, the body burns fatty acids leading to the production of acidic ketones that can be detected by an alcoholic smell on the breath. Hypoglycemic episodes can lead to coma and even death, and is considered