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Abstract Insulin treatment should be introduced in patients with slowly progressive type 1 diabetes (SPIDDM; definite), according to the revised diagnostic criteria of SPIDDM (2023). In contrast, SPIDDM (probable) patients are in a non‐insulin‐dependent state; therefore, a more flexible treatment can be considered, although sulfonylurea agents should be avoided. Insulin treatment has been shown to maintain endogenous insulin secretion capacity in SPIDDM (probable); however, this does not mean that all SPIDDM (probable) patients should use insulin from the early phase. Dipeptidyl peptidase‐4 inhibitors and biguanides might be the treatment of choice for SPIDDM (probable), but no evidence exists for other hypoglycemic agents. In any case, careful monitoring of the endogenous insulin secretion capacity should be carried out, and if a decrease in insulin secretion capacity is suspected, a change in treatment should be considered to prevent progression to an insulin‐dependent state.

Abstract Aims/Introduction This study aimed to identify risk factors that contribute to the progression of slowly‐progressive type 1 diabetes by evaluating the positive predictive value (PPV) of factors associated with the progression to an insulin‐dependent state. Materials and Methods We selected 60 slowly‐progressive type 1 diabetes patients who tested positive for glutamic acid decarboxylase autoantibodies (GADA) at diagnosis from the Japanese Type 1 Diabetes Database Study. GADA levels in these patients were concurrently measured using both radioimmunoassay (RIA) and enzyme‐linked immunosorbent assay (ELISA) techniques. Results Compared with the non‐progressor group (fasting C‐peptide [F‐CPR] levels maintained ≥0.6 ng/mL), the progressor group showed a younger age at diagnosis, lower body mass index (BMI), lower F‐CPR levels and a higher prevalence of insulinoma‐associated antigen‐2 autoantibodies (IA‐2A). The PPV of RIA‐GADA increased from 56.3 to 70.0% in the high titer group (≥10 U/mL), and further increased to 76.9, 84.2, 81.0 and 75.0% when combined with specific thresholds for age at diagnosis

BackgroundThe rate of recurrent spontaneous preterm delivery (sPTD) ranges between 27% and 34% and is 22.3% in Japan. Although it currently remains unclear whether probiotics prevent sPTD, retrospective studies recently reported a reduction in the rate of recurrent sPTD with the administration of probiotics including Clostridium spp., which induce regulatory T cells that play an important role in maintaining pregnancy. ObjectiveThe objective of this trial is to evaluate the preventative effects of available oral probiotics, including Clostridium butyricum, on recurrent sPTD. MethodsThis is a prospective, single-arm, nonblinded, multicenter trial in Japan. The sample size required for this trial is 345 pregnant women with a history of sPTD, considering a clinically significant reduction in the relative risk of 30% (risk ratio=0.7). The primary endpoint is the rate of recurrent sPTD at

Abstract Aims/Introduction Endogenous insulin secretion could be recovered by improving hyperglycemia in patients with type 2 diabetes. This study aimed to investigate the association between short‐term recovery of insulin secretion during hospitalization and clinical background or future glycemic control in patients with type 2 diabetes. Materials and Methods A total of 127 patients with type 2 diabetes were included. The recovery of endogenous insulin secretion was determined using the following indices: index A: fasting C‐peptide index (CPI) at discharge – fasting CPI on admission; index B: postprandial CPI at discharge – postprandial CPI on admission; and index C: Δ C‐peptide immunoreactivity (CPR) (postprandial CPR − fasting CPR) at discharge – ΔCPR on admission. We examined the associations of each index with clinical background and future glycemic control measured by glycosylated hemoglobin and continuous glucose monitoring. Results Using index A and B, the age was significantly younger, whereas BMI and visceral fat area were significantly higher in the high‐recovery group than in the low‐recovery group. Changes in glycosylated hemoglobin levels were significantly greater at 6 and 12 months in the high‐recovery group in the analysis of index C. The receiver operating characteristic curve analysis identified the index B and index C as indicators to predict glycosylated hemoglobin

Abstract The diagnostic criteria for slowly progressive type 1 diabetes (slowly progressive insulin‐dependent diabetes mellitus; SPIDDM) have been revised by the Committee on Type 1 Diabetes of the Japan Diabetes Society. All of the following three criteria must be met for ‘a definitive diagnosis of SPIDDM’: (1) presence of anti‐islet autoantibodies at some point in time during the disease course; (2) absence of ketosis or ketoacidosis at the diagnosis of diabetes with no requirement for insulin treatment to correct hyperglycemia immediately after diagnosis in principle; and (3) gradual decrease of insulin secretion over time, with insulin treatment required at more than 3 months after diagnosis, and the presence of severe endogenous insulin deficiency (fasting serum C‐peptide immunoreactivity

ABSTRACT Pancreatic islet transplantation is a β‐cell replacement therapy for people with insulin‐deficient diabetes who have difficulty in glycemic control and suffer from frequent severe hypoglycemia. However, the number of islet transplantations carried out is still limited in Asia. We report a case of allogeneic islet transplantation in a 45‐year‐old Japanese man with type 1 diabetes. Although the islet transplantation was successfully carried out, graft loss was observed on the 18th day. Immunosuppressants were used in accordance with the protocol, and donor‐specific anti‐human leukocyte antigen antibodies were not detected. Autoimmunity relapse was also not observed. However, the patient had a high titer of anti‐glutamic acid decarboxylase antibody from before the islet transplantation, and autoimmunity might thus have affected the β‐cells in the transplanted islet. The evidence is still scarce to reach conclusions, and further data accumulation is required to enable proper patient selection before islet transplantation.

Abstract Aim/Introduction To investigate the differences in the clinical significance and glutamic acid decarboxylase autoantibody (GADA) affinity between RIA (RIA‐GADA) and ELISA (ELISA‐GADA) in patients with type 1 diabetes. Methods A total of 415 patients with type 1 diabetes were enrolled, including 199 acute‐onset type 1 diabetes, 168 slowly progressive type 1 diabetes (SPIDDM), and 48 fulminant type 1 diabetes. GADA affinity was measured by a competitive binding experiment using unlabeled recombinant human GAD65 protein, and the diagnostic performance of both assays and the relationship between GADA affinity and the decline of fasting C‐peptide (F‐CPR) were examined. Results While the ELISA‐GADA displayed a higher sensitivity than the RIA method in diagnosing type 1 diabetes in acute‐onset patients, about 40% of SPIDDM patients with low‐titer RIA‐GADA were determined as negative by the ELISA method. Patients with type 1 diabetes with RIA‐GADA alone had an older age of onset, less diabetic ketoacidosis, a higher BMI, and a higher F‐CPR compared with patients positive for both RIA‐GADA and ELISA‐GADA. Additionally, 36% of RIA‐GADA‐positive patients had low‐affinity GADA (

Abstract Aims/Introduction This study aimed to investigate the clinical significance and antigen specificity of autoantibodies to insulinoma‐associated antigen‐2 (IA‐2A) by radioimmunoassay (RIA; IA‐2A‐RIA) and enzyme‐linked immunosorbent assay (ELISA; IA‐2A‐ELISA) in Japanese patients with type 1 diabetes. Materials and Methods A total of 338 type 1 diabetic patients were enrolled, including 38 fulminant type 1 diabetes, 168 acute‐onset type 1 diabetes and 137 slowly‐progressive type 1 diabetes (SPIDDM). The concordance, correlation of autoantibody titer, and the relationship between IA‐2A and progression to the insulin‐deficient state were examined. Also, competitive assay was used to examine the antigen specificity. Results The prevalence of IA‐2A‐ELISA was 4–5% lower than that of IA‐2A‐RIA in both the acute‐onset type 1 diabetes and SPIDDM, but the diagnostic sensitivities of both subtypes, when measured in combination with glutamic acid decarboxylase autoantibody, were comparable. The diagnosis of type 1 diabetes using either the RIA or ELISA methods showed substantial agreement with the exponential correlation of autoantibody titers detected by RIA and ELISA. Among the SPIDDM patients, the fasting C‐peptide for IA‐2A‐positive cases by ELISA, but not the RIA method, was significantly lower than in the negative cases (P

Abstract Aims/Introduction This study aimed to identify the clinical factors affecting postoperative residual pancreatic β‐cell function, as assessed by the C‐peptide index (CPI), and to investigate the association between perioperative CPI and the status of diabetes management after pancreatectomy. Materials and Methods The associations between perioperative CPI and clinical background, including surgical procedures of pancreatectomy, were analyzed in 47 patients who underwent pancreatectomy, and were assessed for pre‐and postoperative CPI. The association between perioperative CPI and glycemic control after pancreatectomy was investigated. Results The low postoperative CPI group (CPI

Abstract Aims/Introduction Diastolic cardiac dysfunction in type 2 diabetes (DD2D) is a critical risk of heart failure with preserved ejection fraction. However, there is no established biomarker to detect DD2D. We aimed to investigate the predictive impact of fragmented QRS (fQRS) on electrocardiography on the existence of DD2D. Materials and Methods We included in‐hospital patients with type 2 diabetes without heart failure symptoms who were admitted to our institution for glycemic management between November 2017 and April 2021. An fQRS was defined as an additional R′ wave or notching/splitting of the S wave in two contiguous electrocardiography leads. DD2D was diagnosed according to the latest guidelines of the American Society of Echocardiography. Results Of 320 participants, 122 patients (38.1%) had fQRS. DD2D was diagnosed in 82 (25.6%). An fQRS was significantly associated with the existence of DD2D (odds ratio 4.37, 95% confidence interval 2.33–8.20; p

Abstract Here, we report a case of allogeneic islet transplantation in Japan. A 48‐year‐old man received intraportal islet transplantation (5,945 islet equivalent/kg), and stabilization of blood glucose levels and suppression of hypoglycemia were achieved. In the present case, we used our original assessment method to detect the responses of the recipient’s T cells to islet autoantigens over time to monitor cellular autoimmunity. Other markers could not predict graft dysfunction in advance, but our method detected the activation of islet antigen‐specific CD8+ T‐cell responses before the deterioration of pancreatic β‐cell function, indicating the possibility of the non‐invasive detection of pancreatic β‐cell damage due to recurrent autoimmunity.

Abstract Aims/Introduction Fragmented QRS (fQRS) on electrocardiography is a marker of myocardial fibrosis and myocardial scar formation. This study aimed to clarify the relationship of fQRS with diabetes mellitus and metabolic syndrome (MetS) in Japanese patients. Materials and Methods Approximately 702 individuals who had a routine health checkup at the Hokuriku Health Service Association (Toyama, Japan) in October 2014 were enrolled and categorized into one of the following four groups based on MetS and diabetes mellitus status: with diabetes mellitus (+) MetS+ (164 participants); diabetes mellitus+ without MetS (Mets−; 103 participants); diabetes mellitus− MetS+ (133 participants); and diabetes mellitus− MetS− (302 participants). fQRS was assessed using the results of electrocardiography. Results The prevalence of fQRS was statistically higher in patients with diabetes mellitus+ MetS+ (37%) and diabetes mellitus+ MetS− (35%), than those with diabetes mellitus− MetS+ (14%) or diabetes mellitus− MetS− (10%; P

Abstract Aim/Introduction Insulin administration was found to trigger type 1 diabetes in six Japanese type 2 diabetes patients with type 1 diabetes high‐risk human leukocyte antigen class II and the class I allele of the insulin gene variable number tandem repeat genotype. The objective of the present study was to assess the contribution of non‐human leukocyte antigen single‐nucleotide polymorphisms (SNPs) to the risk of developing insulin‐triggered type 1 diabetes. Materials and Methods We genotyped 13 type 1 diabetes susceptible SNPs in six patients and compared them with those in Japanese controls (Hap Map3‐JPT). The SNPs that showed statistically significant results were further analyzed using non‐diabetic control participants and participants with type 2 diabetes at the Ehime University Hospital. Results The risk allele frequency of BACH2 rs3757247 in the six patients was significantly more frequent than that in 86 Japanese controls (P = 0.038). No significant difference in the allele frequency was observed in the other SNPs. This result was confirmed by the findings that the risk allele frequency of BACH2 in the six patients was significantly higher than that in the non‐diabetic control participants (n = 179) and type 2 diabetes with or without insulin treatment (n = 154 or n = 152; P = 0.035, 0.034 or 0.037, respectively). Despite being statistically not significant, the six patients were all homozygous for the CLEC16A rs12708716 risk allele and five were homozygous for the CLEC16A rs2903692 risk allele. Conclusions In addition to type 1 diabetes high‐risk human leukocyte antigen class II and the class I allele of the insulin gene variable number tandem repeat genotype, the possibility that the risk variants of BACH2 and CLEC16A could contribute to the development of insulin‐triggered type 1 diabetes cannot be excluded.

Abstract A 64‐year‐old woman developed type 1 diabetes 23 years after the diagnosis of idiopathic CD4 lymphocytopenia. To investigate the etiological interaction between idiopathic CD4 lymphocytopenia and type 1 diabetes, we carried out a longitudinal analysis related to islet‐specific autoimmunity. Anti‐glutamic acid decarboxylase antibody had been already weakly positive for at least 16 years and started rising at 6 months before the onset of type 1 diabetes. The seroconversion of anti‐insulinoma‐associated antigen‐2 antibody and insulin autoantibody occurred at the time of onset. The ratio of CD8/CD4 had been gradually increasing for 8 years before type 1 diabetes onset. Notably, islet‐specific glucose‐6‐phosphatase catalytic subunit‐related protein‐reactive CD8+ T cells were detected at type 1 diabetes onset, and the frequency was higher than that in 15 non‐diabetic controls (6.75% vs 0.49 ± 0.78%, mean ± SD). The present type 1 diabetes patient, presented with idiopathic CD4 lymphocytopenia and showed an elevated number of CD8+ T cells, including the islet antigen‐specific CD8+ T cells that might contribute to autoimmune destruction of pancreatic β‐cells.

Abstract Aims/Introduction To evaluate the contribution of pancreatic α‐cell function to the dawn phenomenon, insulin sensitivity, hepatic glucose uptake and glycemic variability in patients with type 1 diabetes. Materials and Methods In 40 patients with type 1 diabetes, arginine stimulation tests were carried out, and the area under the curve (AUC) of glucagon was measured using radioimmunoassays (AUCglcRIA) and enzyme‐linked immunosorbent assays (AUCglcELISA). The ratio of the insulin dose delivered by an artificial pancreas to maintain euglycemia between 04.00 and 08.00 hours or between 00.00 and 04.00 hours was measured as the dawn index. The glucose infusion rate and hepatic glucose uptake were measured using hyperinsulinemic euglycemic clamp and clamp oral glucose loading tests. Glycemic variability in 96 h was measured by continuous glucose monitoring. Results The median dawn index (1.7, interquartile range 1.0–2.8) was not correlated with AUCglcRIA (R2 = 0.03, P = 0.39) or AUCglcELISA (R2 = 0.04, P = 0.32). The median glucose infusion rate (7.3 mg/kg/min, interquartile range 6.4–9.2 mg/kg/min) was significantly correlated with AUCglcRIA (R2 = 0.20, P = 0.02) and AUCglcELISA (R2 = 0.21, P = 0.02). The median hepatic glucose uptake (65.3%, interquartile range 40.0–87.3%) was not correlated with AUCglcRIA (R2 = 0.07, P = 0.26) or AUCglcELISA (R2 = 0.26, P = 0.79). The standard deviation of glucose levels measured by continuous glucose monitoring was significantly correlated with AUCglcRIA (R2 = 0.11, P = 0.049), but not with AUCglcELISA (R2 = 0.01, P = 0.75). Conclusions Pancreatic α‐cell function contributed to insulin sensitivity in patients with type 1 diabetes.

Objectives. A single-arm prospective study was conducted among Japanese patients with type 2 diabetes having preserved ejection fraction. The aim was to investigate (1) whether liraglutide therapy could improve B-type natriuretic peptide (BNP) levels and diastolic cardiac function assessed by the E-wave to E′ ratio (E/E′) using transthoracic echocardiography (TTE), and (2) whether E/E′ contributed to BNP improvement independent of bodyweight reduction (UMIN000005565). Methods. Patients with type 2 diabetes and left ventricular ejection fraction LVEF≥40% without heart failure symptoms were enrolled, and daily injection with liraglutide (0.9 mg) was introduced. Cardiac functions were assessed by TTE before and after 26 weeks of liraglutide treatment. Diastolic cardiac function was defined as septal E/E′≥13.0. Results. Thirty-one patients were analyzed. BNP and E/E′ improved, with BNP levels declining from 36.8±30.5 pg/mL to 26.3±25.9 pg/mL (p=0.0014) and E/E′ dropping from 12.7±4.7 to 11.0±3.3 (p=0.0376). The LVEF showed no significant changes. E/E′ improved only in patients with E/E′≥13.0. Favorable changes in E/E′ were canceled when adjusted for body mass index (BMI). Multivariate linear regression analysis revealed that the left ventricular diastolic diameter and ∆E/E′/∆BMI contributed to ∆BNP/baseline BNP (p=0.0075, R2=0.49264). Conclusions. Liraglutide had favorable effects on BNP and E/E′ but not on LVEF. E/E′ improvement was only seen in patients with diastolic cardiac function. Body weight reduction affected the change of E/E′. The BMI-adjusted E/E′ significantly contributed to the relative change of BNP. GLP-1 analog treatment could be considered a therapeutic option against diabetic diastolic cardiac dysfunction regardless of body weight. This trial is registered with the University Hospital Medical Information Network in Japan, with clinical trial registration number: UMIN000005565.

A major problem after clinical autologous islet transplantation (AIT) is the difficulty in achieving insulin independence. To follow up on our demonstration in a murine model that high-mobility group box 1 (HMGB1) was released from islets and involved in early loss of transplanted islets, we tested the role of HMGB1 in clinical AIT. Serum HMGB1 levels from 15 AIT patients were significantly elevated during islet infusion (7.6 ± 1.2 ng/ml) and 24 h after infusion (8.0 ± 1.4 ng/ml) compared to admission levels (2.4 ± 0.6 ng/ml). The first elevation of HMGB1 was associated with islet damage, but the later elevation was not. The change in the HMGB1 level from admission to first peak (ΔHMGB1) was significantly higher in the AIT group (8.1 ± 1.1 ng/ml) than in the pancreatectomy-only control (2.2 ± 0.5 ng/ml) ( p < 0.05). Circulating serum levels of soluble receptor for advanced glycation end products (sRAGE) were also elevated during islet infusion. In vitro studies demonstrated that damaged human islets released HMGB1 but not sRAGE. In terms of outcomes, the insulin-free group showed significantly lower ΔHMGB1 (5.2 ± 0.6 ng/ml) and higher ΔsRAGE (2.3 ± 0.6 ng/ml) than the insulin-dependent group (10.6 ± 1.9 ng/ml and 0.7 ± 0.2 ng/ml, respectively). The ΔHMGB1 correlated with the number of white blood cell, IP-10, EGF, and eotaxin. In conclusion, serum HMGB1 was elevated in AIT and could be associated with inflammatory reactions that deteriorate islet engraftment. Therefore, anti-HMGB1 therapy might be a candidate for further improving the outcomes of clinical AIT.

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4(+) T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4(+) T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4(+) T cells belonged to the CD45RO(+) memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4(+) T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4(+) T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4(+) T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.

Islet transplantation is one of the most promising therapies for type 1 diabetes (T1D). A major issue in islet transplantation is the loss of graft function at late phase. Several studies suggested the involvement of islet-specific T-cells in such islet graft dysfunction. In this study, we investigated the breadth and type of glutamic acid decarboxylase 65 (GAD65)-specific T-cells in T1D patients after allogeneic islet transplantation. Peripheral blood mononuclear cells (PBMCs) were obtained from islet-transplanted T1D patients during insulin-independent period and cultured for 7 days with pools of GAD65 overlapping peptides in the presence of IL-2. Cytokine secretion profiles of peptide-reactive T-cells were analyzed after a short-term restimulation with the same peptides by a multiplex bead-based cytokine assay and by an intracytoplasmic cytokine detection assay. Robust GAD65-specific CD4 + and CD8 + T-cell responses were detected in patients who eventually developed chronic graft dysfunction. Multiple GAD65 peptides were found to induce specific T-cell responses in these patients, indicating that the repertoire of GAD65-specific T-cells was broad. Furthermore, GAD65-specific CD4 + T-cells were composed of heterogeneous populations, which differentially expressed cytokines including IFN-γ and type 2 cytokines, but not IL-10. In contrast, patients who showed only marginal GAD65-specific T-cell responses maintained substantially longer graft survival and insulin independence. In conclusion, our study suggests that the emergence of islet-specific T-cells precedes the development of chronic graft dysfunction in islet-transplanted patients. Thus, our observations support the hypothesis that these islet-specific T-cells contribute to the development of chronic islet graft dysfunction.

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.

One of the major issues in clinical islet transplantation is the poor efficacy of islet isolation. During pancreas preservation and islet isolation, islets suffer from hypoxia as islets are highly sensitive to hypoxic conditions. Cold preservation has been applied to minimize hypoxia-induced cell damage during organ preservation. However, the studies related to hypoxia-induced islet cell damage during islet isolation are limited. Recently, we demonstrated that mouse islets contain high levels of high-mobility group box 1 protein (HMGB1), and during proinflammatory cytokine-induced damage, islets release HMGB1 outside the cell. The released HMGB1 is involved in the initial events of early islet loss. In the present study, we hypothesize that low temperature conditions could prevent both hypoxia induced islet cell damage and HMGB1 release from islets in a mouse model. Isolated mouse islets underwent normoxic condition (95% air and 5% CO 2 ) at 37°C or hypoxic conditions (1% O 2 , 5% CO 2 , and 94% N 2 ) at 37°C (hypoxia-37°C islets), 22°C (hypoxia-22°C islets), or 4°C (hypoxia-4°C islets) for 12 h. In vitro and in vivo viability and functionality tests were performed. HMGB1, IL-6, G-CSF, KC, RANTES, MCP-1, and MIP-1 α levels in the medium were measured. Low temperature conditions substantially reduced hypoxia-induced necrosis ( p < 0.05) and apoptosis ( p < 0.05). In addition, low temperature islet culture significantly increased the insulin secretion from islets by high glucose stimulation ( p < 0.05). All of the recipient mice reversed diabetes after receiving the hypoxia-4°C islets but not after receipt of hypoxia-37°C or 22°C islets. The amounts of released HMGB1, IL-6, G-CSF, KC, RANTES, MCP-1, and MIP-1 α were significantly reduced in the hypoxia-4°C islets compared to those of the hypoxia-37°C islets ( p

Autologous islet transplantation after total pancreatectomy is an excellent treatment for painful chronic pancreatitis. Traditionally, islets have been isolated without purification; however, purification is applied when the tissue volume is large. Nevertheless, the impact of tissue volume and islet purification on clinical outcomes of autologous islet transplantation has not been well examined. We analyzed 27 cases of autologous islet transplantation performed from October 2006 to January 2011. After examining the relationship between tissue volume and portal pressure at various time points, we compared islet characteristics and clinical outcomes between cases with complications (complication group) and without (noncomplication group), as well as cases with purification (purification group) and without (nonpurification group). Tissue volume significantly correlated with maximum ( R = 0.61), final ( R = 0.53), and delta (i.e., difference between base and maximum; R = 0.71) portal pressure. The complication group had a significantly higher body mass index, tissue volume, islet yield, and portal pressure (maximum, final, delta), suggesting that complications were associated with high tissue volume and high portal pressure. Only one of four patients (25%) in the complication group became insulin free, whereas 11 of 23 patients (49%) in the noncomplication group became insulin free with smaller islet yields. The purification group had a higher islet yield and insulin independence rate but had similar final tissue volume, portal pressure, and complication rates compared with the nonpurification group. In conclusion, high tissue volume was associated with high portal pressure and complications in autologous islet transplantation. Islet purification effectively reduced tissue volume and had no negative impact on islet characteristics. Therefore, islet purification can reduce the risk of complications and may improve clinical outcome for autologous islet transplantation when tissue volume is large.

For clinical islet transplantation, isolated islets deteriorate rapidly in culture, although culturing islets prior to transplantation provides flexibility for evaluation of isolated islets and pretreatment of patients. In the present study, we compared human fresh islets to cultured islets with in vitro and in vivo assays. After culture for 24, 48, and 72 h, islet yield significantly decreased from 2,000 to 1,738 ± 26 (13% loss), 1,525 ± 30 (24% loss), or 1,298 ± 18 IEQ (35% loss), respectively. The ATP contents were significantly higher in the 6-h cultured group (near fresh group) than in 48-h culture groups. The stimulation index was relatively higher in the 6-h cultured group than in 48-h cultured group. Human islets with or without culture were transplanted into diabetic nude mice. The attainability of posttransplantation normoglycemia was significantly higher in fresh group than in the culture groups. Intraperitoneal glucose tolerance testing (IPGTT) showed that the blood glucose levels of mice transplanted with fresh islets were significantly lower than with cultured islets at 30, 60, 90, and 120 min after injection. These data suggest that human islet transplantation without culture could avoid the deterioration of islets during culture and improve the outcome of islet transplantation. Based on these data, we have transplanted fresh islets without culture for our current clinical islet transplantation protocol.

Porcine islets are a promising resource for xenotransplantation. However, low efficacy of islet isolation because of their marked fragility remains a problem. Recently we found that the standard purification method using COBE 2991 cell processor (COBE) with Ficoll density gradient solution damaged islets mechanically by high shearing force. In this study, we evaluated our new purification method using large plastic bottles for the efficacy of islet purification. Ten porcine pancreata were used. The average warm ischemic time was over 40 min; therefore, these pancreata were considered to be in a marginal condition. After digestion, the digested tissue was divided into three groups. Each group was purified using either top loading method with bottle (top group) or bottom loading method with bottle (bottom group) or standard COBE method (COBE group). Islet yield per pancreas weight (IEQ/g) and the rate of postpurification recovery in the top group were significantly higher than the COBE group (top: 8060 ± 1652 IEQ/g, bottom: 4572 ± 614 IE/g, COBE: 3900 ± 734 IE/g. p < 0.02 in top vs. COBE; top percentage of recovery: 99.3 ± 12.3%, bottom: 62.6 ± 8.8%, COBE: 49.5 ± 6.7%, p < 0.02 in top vs. bottom and COBE). The average sizes of purified islets in the top and bottom groups were significantly larger than COBE group (Average diameter top: 156 ± 8 μm, bottom: 147 ± 6 μm, COBE: 119 ± 6 μm, p < 0.01 in top vs. COBE and in bottom vs. COBE), which indicated that bottle method can reduce shear force during purification. Our new purification using top loading bottle method enabled us to obtain a high yield of porcine islets from marginal pancreata.

For islet transplantation, maintaining organ viability after pancreas procurement is critically important for optimal graft function and survival. We recently reported that islet yield was significantly higher in the modified ET-Kyoto (MK) solution, which includes a trypsin inhibitor (ulinastatin), compared with the UW solution, and that the advantages of MK solution are trypsin inhibition and less collagenase inhibition. In this study, we compared ulinastatin with other trypsin inhibitors, gabexate mesilate, and nafamostat mesilate, in preservation solution for islet isolation. Ulinastatin was easily dissolved in ET-Kyoto solution, while ET-Kyoto with gabexate mesilate and nafamostat mesilate became cloudy immediately after addition. Although there were no significant differences in islet yield among the three groups, viability was significantly higher for the MK group than for the GK group or the NK group. The stimulation index was significantly higher for the MK group than for the GK group. In summary, there are no other trypsin inhibitors that are more effective than ulinastatin. Based on these data, we now use ET-Kyoto solution with ulinastatin for clinical islet transplantation.

For pancreatic islet transplantation, the most common method of islet purification is density gradient centrifugation because of the differences in density between islets and acinar tissue. The density of islets/acinar tissue depends on several conditions, such as osmolality of purification solution. In this study, we evaluated the osmolality of iodixanol-controlled density gradients (400, 450, and 500 mOsm/kg) on the islet purification step. The density of the purification solutions was controlled by changing the volumetric ratio of iodixanol and the purification solutions (iodixanol-Kyoto solutions; IK solutions). The osmolality of density gradients was controlled by addition of 10x Hanks balanced salt solution (HBSS) solution. Density of both islets and acinar tissue increased relative to increase of the osmolality of purification solutions. There were no significant differences among the three groups on islet yield after density-adjusted purification and the rate of postpurification recovery. In vitro and in vivo assays suggest that the quality of islets was similar among the three groups. Our data suggest that efficacy of purification and quality of isolated islets is similar when the osmolality of purification solutions is between 400 and 500 mOsm/kg and density adjustment is applied. Since the density of islet and acinar tissue is changed according to osmolality, the density adjustment is important when using several osmolality solutions.

Porcine islets are considered to be a promising resource for xenotransplantation. However, it is difficult to isolate porcine islets because of the marked fragility and rapid dissociation. Endogenous trypsin is one of the main factors to damage islets during the isolation procedure. Recent studies have suggested that trypsin inhibitors during the preservation of pancreas or the collagenase digestion can improve the result of islet isolation. In this study, we examined whether a 1-antitrypsin (Aralast™), which inhibits several endogenous proteases and has immunomodulatory properties, can protect islets from the proteases and improve the results of porcine islet isolation. Twelve porcine pancreata were divided into three groups: without Aralast group (standard, n = 5), preserved with Aralast using the ductal injection (DI) method (DI, n = 3), and with Aralast using the DI method and in the collagenase solution (DI+C, n = 4). Efficacy of islet isolation was assessed by islet yields, purity, and viability. The trypsin activity of the preservation and the digestion solution during the isolation procedure was measured. During islet isolation, the trypsin activity in DI+C group was significantly inhibited compared to the standard group, whereas DI group showed less effect than DI+C group. The average of postpurification islet equivalents (IEQ) per pancreas weight in the DI+C group was significantly higher than the standard group (standard: 3516 ± 497 IEQ/g, DI: 4607 ± 1090 IEQ/g, DI+C: 7097 ± 995 IEQ/g; p = 0.017 between standard and DI+C). In the DI+C group, stimulation index was higher than in other groups, although there was no significant difference. The presence of Aralast in both DI solution and collagenase solution markedly inhibited trypsin activity during pancreas digestion procedure and improved the porcine islet isolation. Inhibition of trypsin activity by Aralast could improve porcine islet isolation.

Islet transplantation is one of the most promising treatments for an unstable form of type 1 diabetes. However, islet transplantation still has some obstacles, such as low success rate of islet isolation, difficulty to obtain long-term insulin freedom, and adverse events related to transplant protocol. We describe the adverse events of current clinical islet transplantation at our institute in this report. Nine type 1 diabetic patients received 17 islet infusions from March 2005 to October 2008. The islet infusion procedure and immunosuppression regimen were based on a modified Edmonton protocol. Severe adverse events (SAEs) were defined as events that were more than grade 3 according to the Terminology Criteria for Adverse Events in Trials of Adult Pancreatic Islet Transplantation, version 4.1 (Collaborative Islet Transplant Registry, CITR). Sixteen events were reported as SAEs and among them 12 events were probably or definitely related to transplant protocols; all occurred within 1 year after infusion except for one. Five adverse events (31%) occurred within 10 days after transplantation and were related to infusion procedures. Seven events (44%) occurred after 50 days and were related to immunosuppressive therapy. SAEs related to the protocol included three events of elevated liver enzymes, two of hemorrhage into gall bladder or peritoneal cavity, two of neutropenia, two of infection, one of vomiting, one of diarrhea, and one of renal dysfunction. All events were grade 3, except for one case that was grade 4 of neutropenia. All SAEs resolved with no sequelae. Neoplasms and deaths were not observed in our study. The present study suggests need to improve both infusion procedure and immunosuppressive strategy from the view of preventing SAEs.

Total or partial pancreatectomy followed by autologous islet transplantation is a therapeutic option for the treatment of refractory chronic pancreatitis (CP). Maximization of islet yields from fibrotic and inflamed organs is crucial for prevention of posttransplant diabetes. We adapted technical advancements developed for islet allotransplantation toward islet autotransplantation. Eight patients (two men, six women; ages 24–58 years) underwent total ( n = 7) or partial ( n = 1) pancreatectomy for the treatment of CP refractory to maximal medical management. Pancreata were preserved in UW solution (UW group) in initial three cases and the last five pancreata were preserved with pancreatic ductal injection followed by ET-Kyoto/oxygenated PFC solutions (DI+TLM group). Islets were isolated by modified Ricordi method and were purified only in one case. All islet infusions were performed under general anesthesia via direct vein injection into the portal venous system with pressure monitoring. Total islet yields (129,314 ± 51,627 vs. 572,841 ± 116,934 IEQ, p < 0.04), islet yield/pancreas weight (1,233 ± 359 vs. 6,848 ± 847 IEQ/g, p < 0.003), and islet yield/patient body weight (1,951 ± 762 vs. 7,305 ± 1,531 IEQ/kg, p < 0.05) were significantly higher in the DI+TLM group when compared to the UW group. Pellet size was also higher (5.3 ± 0.3 vs. 13.5 ± 3.4 ml) in the DI+TLM group, suggesting that this method of preservation effectively protected pancreatic tissue against autolysis. First month posttransplant basal C-peptide and the secretory unit of islet transplant objects (SUITO) index were also higher in the DI+TLM group when compared to the UW group (2.0 ± 0.3 vs. 1.4 ± 0.4 ng/ml and 42.6 ± 12.7 vs. 14.6 ± 5.6, respectively). There were no technical complications related to the infusion. Our results suggest that higher islet yields can be achieved even from chronically inflamed and fibrotic organs using DI+TLM. The techniques applied for islet isolations from normal pancreata are showing promise for fibrotic pancreata from CP patients.

One endpoint of clinical islet cell transplantation for type 1 diabetic patients is the elimination or reduction of hypoglycemia. We previously developed a simple tool to evaluate islet graft function: the secretory unit of islet transplant objects (SUITO) index. The aim of this study is to clarify the association between the SUITO index and hypoglycemic episodes. Data from 310 clinical evaluations of 11 islet recipients were included in this study. Fasting plasma C-peptide and glucose levels were measured at every evaluation. The SUITO index was calculated according to the following formula: 1500 × C-peptide level (ng/ml)/[blood glucose level (mg/dl) – 63]. The number of hypoglycemic events (

Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions ( N = 3) and the second protocol only required a single infusion ( N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol enabled us to perform successful single-donor islet transplantations. Further large-scale studies are necessary to confirm these results and clarify the mechanism of each component.

Total pancreatectomy with autologous islet cell transplantation (TP with AIT) is an effective treatment for chronic pancreatitis patients with severe abdominal pain. Body mass index (BMI) of the pancreatic donor is proven to be a useful predictor for islet isolation and transplantation outcomes in allogenic islet transplantation. However, the association between BMI and islet isolation outcome and/or metabolism after AIT was previously unclear. Twelve patients who received TP with AIT at our hospital were included in this study. All pancreata were preserved with both pancreatic ductal injection and oxygen-charged static two-layer method using ET-Kyoto solution. The cohort was divided into two groups: low BMI group (BMI < 23 kg/m 2 , n = 5) and high BMI group (BMI ≥ 23, n = 7). The high BMI group had a significantly higher islet yield per gram than the low BMI group both in pancreas postdigestion and in final product (postdigestion: 7330 ± 539 vs. 3509 ± 563 IE/g; p < 0.001; final product: 6555 ± 585 vs. 3476 ± 546 IE/g; p = 0.004). For islet yield in final product per patient body weight, the high BMI group also had significantly higher islet yield than the low BMI group (7997 ± 779 vs. 4175 ± 750 IE/kg, p = 0.007). Insulin independence rate in the high BMI group (71%) was also higher than that low BMI group (40%), but it did not reach statistical significance. Pancreata from patients with higher BMI could obtain higher islet yield in the setting of autologous islet cell transplantation for chronic pancreatitis.

β-Cell replacement therapy via islet transplantation is an effective treatment for diabetes mellitus, but its widespread use is severely limited by the shortage of donor organs. Because pancreatic stem/progenitor cells are abundantly available in the pancreas of these patients and in donor organs, the cells could become a useful target for β-cell replacement therapy. We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we used the techniques to identify and isolate human pancreatic stem/progenitor cells. The cells from a duct-rich population were cultured in 23 kinds of culture media, based on media for mouse pancreatic stem cells or for human embryonic stem cells. The cells in serum-free media formed “cobblestone” morphologies, similar to a mouse pancreatic stem cell line. On the other hand, the cells in serum-containing medium and the medium for human embryonic stem cells formed “fibroblast-like” morphologies. The cells divided actively until day 30, and the population doubling level (PDL) was 6–10. However, the cells stopped dividing after 30 days in any culture conditions. During the cultures, the nucleus/cytoplasm (N/C) ratio decreased, suggesting that the cells entered senescence. Exendin-4 treatment and transduction of PDX-1 and NeuroD proteins by protein transduction technology into the cells induced insulin and pancreas-related gene expression. Although the duplications of these cells were limited, this approach could provide a potential new source of insulin-producing cells for transplantation.

Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 ± 64,319 vs. 354,836 ± 89,649 IE, p < 0.01) and viability (97.3 ± 1.2% vs. 92.6 ± 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.