Introduction: Allogeneic islet cell transplantation (ICT) is a promising treatment for patients with brittle type 1 diabetes. Recent reports have demonstrated that T cell depletiing immunosuppression protocol could improve islet engraftment as well as prolong graft function. We have implemented the T cell depletion plus antiinflammatory (TCD-AI) protocol using antithymocyte globulin (ATG) and double blockage of IL-1β and TNF-α. Global investigation of peripheral blood transcriptions is helpful for understanding systemic responses as well as discovering diagnostic biomarkers. Modular level analysis for cDNA microarray allows effective interpretation on the background determination with multiple immunologic disease populations (1). In this study, peripheral blood transcriptions were measured to explore systemic biological response for early phase of ICT with TCD-AI protocol. Methods: Five islet recipients received TCD-AI protocol with ATG for induction, along with anakinra and etanercept. Peripheral blood samples were collected on the screening day (baseline), post-transplant days 4, 7, 14, 28 and 42. Isolated RNA were hybridized on to Illumina HumanHT-12 v4 BeadChip. Probe-level differential gene expression analysis was conducted using mixed model adjusting for correlation for longitudinal data to compare between baseline and post-transplant samples. A false discovery rate of 0.1 was used to account for multiple testing. Modular analysis of gene expressions were employed with previously determined sets of genes (1). Results: Four patients (80%) achieved insulin independence after single islet infusion and the median duration of insulin independence is 17.5 months (range: 4-36 months) as of the analysis. The number of genes that were expressed with significant difference (p< 0.05) are 866, 465, 492, 160, 353 and 80 on day 4, 7, 14, 21, 28 and 42 after transplant when compare to baseline. Modular analysis of cDNA microarray on day 4 revealed upregulated modules annotated as interferon responses (M3.4), inflammation (M4.2), myeloid lineage (M4.6) and cell cycle (M6.13), and downregulated modules as coagulation cascade (M3.6), T-cells (M4.1 and M6.15), lymphoid lineage (M4.7 and M6.9) and cytotoxicity (M4.15) (Figure 1). Among these modules, T-cells (M4.1 and M6.19) and cytotoxicity (M4.15) modules were downregulated throughout the study period.[Figure 1]Figure 1. Blood transcriptional fingerprints with islet recipients receiving TCD-AI protocol. Relative changes in whole blood gene expressions with islet recipients for day 4 to 42, compared to baseline. Colored spots represent the percentage of significantly upregulated (red) or downregulated (blue) transcripts (p< 0.002). Conclusion: Gene expression modules annotated as T-cells and cytotoxicity were downregulated in early phase of ICT, suggesting that effective control of cellular immune response as the background mechanism of improved outcome of TCD-AI protocol. (1) Chaussabel D, et al. Immunity 2008;29:150-164.

RESEARCH DESIGN AND METHODS—Eleven islet recipients were included in this study. The patients visited our clinic monthly after ICT and provided blood samples for fasting C-peptide (n = 270), which were used to evaluate islet graft function. They also provided their SMBG data through an automatic data collection system. The SMBG data for 3 days immediately before eachclinic visit were evaluatedusing the following assessments:M value, meanamplitude of glycemic excursions, J index, index of glycemic control, average daily risk range, and glycemic risk assessment diabetes equation. The cluster analysis was performed for both SMBG assessments and samples. Multivariate logistic regression analysis was used to evaluate the clusters of SMBG for assessing islet graft function. RESULTS—Analysis for SMBG assessments revealed five types of clusters, which showed similar patterns according to functional or dysfunctional islet graft phase. Two clusters, the euglycemiacluster(P,0.001)andthehypoglycemiacluster(P=0.001),weresignificantfactors in the logistic model for islet graft function. The SMBG clusters had significant correlations with clinical graft indexes (P , 0.001). CONCLUSIONS—Cluster analysis of SMBG data as part of an automated data quality system could allow discrimination of islet graft dysfunction after ICT. This approach should be considered for islet recipients. Diabetes Care 34:1799–1803, 2011

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.

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/H20; 400 group, 500 group, and 600 group, respectively). Each tube was gently shaken at 4 degrees 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)0, 15 minutes) could release islets from surrounded exocrine tissue.

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(7), 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.

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.