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.

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.

Association between the secretory unit of islet transplant objects index and satisfaction with insulin therapy among insulin-dependent islet recipients.

INTRODUCTION: When patients do not become insulin independent after islet cell transplantation (ICT), another aim is to eliminate severe hypoglycemia. Previously we reported that a secretory unit of islet transplant objects (SUITO) index score >10 was associated with a reduction of severe hypoglycemia. In this study, we assessed patients' satisfaction with their insulin therapy based on the SUITO index. METHODS: The study involved 11 islet recipients with type 1 diabetes who underwent ICT but still used insulin. From those patients, 41 Insulin Therapy Satisfaction Questionnaires (ITSQ) were collected. The SUITO index (fasting C-peptide [ng/mL] × 1500/blood glucose [mg/dL] - 63) was calculated at the same outpatient visits that the survey was administered. ITSQ scores were summarized using subscales and compared among 3 groups: the pre-ICT group, the low-SUITO group (SUITO index score <10 post-ICT), and the high-SUITO group (SUITO index score ≥10). Higher survey scores indicated better satisfaction. RESULTS: Significant trend relationships across the 3 groups were observed in the ITSQ total score (P = .02 with Jonckheere-Terpstra test) and subscale scores of glycemic control (P < .001), hypoglycemic control (P = .01), and inconvenience of regimen (P = .004). The pairwise comparisons between the 3 groups found significant differences: high SUITO versus both pre-ICT and low SUITO for the total ITSQ score (P = .03 and .005, respectively) and glycemic control score (P = .008 and .001, respectively), and high SUITO versus low SUITO for hypoglycemic control score (P = .04) and inconvenience of regimen score (P = .008). CONCLUSION: Islet recipients with a SUITO index ≥10 experienced higher satisfaction with insulin injection therapy compared with the pre-ICT group, even though they were insulin dependent. A SUITO index ≥10 is a reasonable benchmark for successful ICT.

Assessment of human islet isolation with four different collagenases.

BACKGROUND: The isolation of islets from the human pancreas critically depends on the efficiency of the digestive enzymes. Liberase HI had been used as a standard preparation until the issues concerning bovine spongiform encephalopathy. Thus, we must now use other collagenases for clinical islet transplantation, four of which we have evaluated herein. METHODS: The digestion of each of 17 pancreata from brain-dead donors was performed using the following collagenases: Liberase HI (HI; Roche, n = 9); Liberase MTF C/T (MTF; Roche, n = 4); Collagenase NB1 Premium Grade (NB1; Serva, n = 7); or Clzyme Collagenase HA (CI, VitaCyte, n = 4). Islet isolations were based on the Edmonton protocol for HI, whereas our modified islet isolation method was used for the three new enzymes (MTF, NB1, and CI). RESULTS: There were no significant differences in donor age, body mass index, pancreas size, and cold ischemic time among the four groups. The phase I time in the NB1 group was significantly shorter than in the CI group (P = .0014). The prepurification IEQ/g in the HI group was significantly lower than the others (P = .0003 vs MTF, .0007 vs NB1, and .0009 vs CI, respectively). The postpurification IEQ/g in the MTF group was significantly higher than in the HI group (P = .006). The viability in the NB1 group was significantly greater than the HI group (P = .003). CONCLUSION: Three new enzymes (MTF, NB1, and CI) may enable us to obtain higher islet yields than with HI.

Excellence of suito index for assessing clinical outcome of islet transplantation.

BACKGROUND: Monitoring functional islet mass after transplantation is critical to follow patients. Previously we demonstrated that the average secretory unit of islet transplant objects (SUITO) index within 1 month was an excellent predictor of insulin-free status or reduction in insulin dose. In this study, we analyzed the usefulness of daily SUITO index to assess clinical outcomes. METHODS: Five patients underwent islet transplantation, including 3 who received 2 transplantations and 2 who received a single graft. All 5 patients achieved insulin-free status with 3 remaining insulin free at the time of evaluation. We analyzed the daily relative insulin dose and SUITO index. The daily relative insulin dose was calculated as the total daily insulin dose/average pretransplant insulin dose. The SUITO index was calculated as [fasting C-peptide (ng/mL)]/[fasting blood glucose (mg/mL) - 63] x 1,500. The data analyzed based on time after islet transplantation were categorized as within or after 1 month. RESULTS: Within 1 month after islet transplantation, there was no correlation between the daily relative insulin dose and the daily SUITO index (P = .068; R = -0.33). After 1 month, the daily relative insulin dose and the daily SUITO index were strongly correlated (P < .0001; R = -0.70). When the cutoff value of the SUITO index was decided at 26 for insulin-free status, the positive predictive value was 84.1% and negative predictive value 89.4%. CONCLUSION: SUITO index was an excellent index to assess clinical outcomes beyond 1 month after islet transplantation.

Secretory unit of islet transplant objects (SUITO) index can predict outcome of intravenous glucose tolerance test.

BACKGROUND: Simple monitoring of engrafted islet function is important for follow-up of recipients after islet transplantation. We previously developed a simple assessment tool for islet graft function; the secretory unit of islet transplant objects (SUITO) index. The aim of this study was to clarify the relationship between the SUITO index and the outcomes of intravenous glucose tolerance tests (IVGTT). METHODS: Fifteen series of blood samples from 6 islet recipients were collected before 3, 5, 10, 20, and 30 minutes after injection of 0.5 g/kg 50% dextrose. The SUITO index was calculated using plasma C-peptide and glucose level at fasting baseline. Samples were divided into the following 3 groups; low-SUITO (SUITO index <10; n = 3); middle-SUITO (SUITO index > or =10 to <26; n = 4); and high-SUITO (SUITO index > or =26; n = 8). RESULTS: A threshold SUITO index of 26 showed good sensitivity (85.7%) and specificity (75.0%) to predict a blood glucose level of >10 mmol/L at 30 minutes. Blood glucose levels in the low-SUITO group were significantly higher than among the other 2 groups at baseline and 10, 20 and 30 minutes (P < .05). Glucose-level areas under the receiver-operating characteristic curve during IVGTT in the low-SUITO group were also significantly larger than among the other 2 groups (P < .05). CONCLUSION: The SUITO index, using only a fasting blood sample, predicted IVGTT outcomes.

Improved method of human islet isolation for young donors.

BACKGROUND: Although islet transplantation using young donors is more effective than older donors, islet isolation from young donor is notoriously difficult. This may relate to islet ontogeny and collagen composition in the young pancreas. Therefore, we examined whether a high concentration of collagenase could improve the separation of islets from exocrine tissues resulting in an high islet yield. METHODS: We used six human pancreata from brain-dead donors of less than 30 years old. Islet isolation was performed based on the Edmonton protocol with modifications. All pancreata were digested with Collagenase NB1 Premium Grade (Serva). The pancreas was expanded by injecting either 200 mL of cold collagenase solution (2.5 mg/mL, standard group, n = 3) or 100 mL of solution (5 mg/mL, new group, n = 3) in a controlled manner under low pressure for 5 minutes. Then the pressure was raised for another 5 minutes. The following procedure and evaluation were performed based on the Edmonton protocol. RESULTS: Phase II time in the new group was significantly shorter than the standard group. The ratio of embedded islets in the new group was significantly lower than the standard group. The postpurification islet equivalents per pancreas weight (IEQ/g) and the recovery rate in the new group were higher than the standard group, but not significantly. There was no significant difference in the postpurification purity, viability, and final tissue volume. CONCLUSION: Our simple modification with an initially concentrated collagenase preparation using a syringe significantly improved the ratio of embedded islets, resulting in a higher yield from young donors.

Induction of insulin-producing cells from human pancreatic progenitor cells.

INTRODUCTION: We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions. MATERIALS AND METHOD: Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein. RESULTS: The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes. CONCLUSION: Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.

Comparison of fresh and cultured islets from human and porcine pancreata.

INTRODUCTION: For clinical islet transplantation, many centers have recently introduced of human islet cultures prior to transplantation. They provide flexibility to evaluate isolated islets and pretreat patients. However, isolated islets deteriorate rapidly in culture. In the present study, we compared fresh human and porcine islets with cultured islets for c-Jun NH(2)-terminal kinase (JNK) activity. MATERIALS AND METHODS: Islet isolations from human and porcine pancreata were performed using the standard Ricordi technique with a modified Edmonton protocol. Isolated islets cultured for 24 hours at 37 degrees C with 5% CO(2) in culture medium were evaluated for counts and JNK activity. RESULTS: After 24 hours of culture, the percentages of surviving islets were 86.9% for human and 47.3% for porcine sources. JNK activity in isolated islets declined to a low baseline level after 24-hour culture. CONCLUSION: Both human and porcine islets deteriorated rapidly in 24-hour cultures, although the in vitro conditions did not induce JNK activation.

Microvascular ischemia in patients with myotonic dystrophy.

Two women with myotonic dystrophy underwent dipyridamole thallium-201 (201Tl) myocardial perfusion imaging, after which one patient developed flat T waves in lead I and aV(L), and inverted T waves in leads V(2-6). The other patient developed a nonspecific intraventricular block that progressed to complete left bundle branch block and was associated with chest discomfort. Reversible scintigraphic defects were observed in both women. Although there was evidence that suggested myocardial ischemia on the ECG changes and 201Tl scintigraphic findings, coronary angiography demonstrated no significant stenoses in either patient. These findings suggest that microvascular dysfunction may lead to myocardial ischemia and conduction disturbances in patients with myotonic dystrophy.