[Improvement of tissue fibrosis in TAFRO syndrome by prednisolone monotherapy].

We report the case of a 48-year-old man who presented with fatigue and weight loss. A local physician observed elevated alkaline phosphatase levels, anemia, thrombocytopenia, and renal dysfunction. Fever also appeared, and the patient was admitted to our hospital. Computed tomography revealed hepatosplenomegaly, pleural and ascitic fluid, and left axillary lymphadenopathy. Bone marrow biopsy indicated hyperplasia with increased megakaryocytes and reticulin fibrosis. Axillary lymph node biopsy showed Castleman's disease-like features. Liver biopsy revealed proliferation of reticulin fibrosis. Therefore, TAFRO syndrome was diagnosed and treatment with 1 mg/kg prednisolone was started. Anemia and thrombocytopenia improved, and after 24 weeks of treatment, serum hyaluronic acid and type IV collagen decreased to the normal range. Bone marrow biopsy after 18 weeks of treatment showed decreased reticular fibers. In TAFRO syndrome, improvement of liver and bone marrow fibrosis can be expected with adequate intervention, and serum hyaluronic acid and type IV collagen are useful for evaluating fibrosis.

[A Case of Immune-Related Adverse Events Affecting the Lungs, Skin, and Pituitary Gland during Pembrolizumab Treatment].

The patient was a 69-year-old man diagnosed with stage ⅣB lung adenocarcinoma with 95% programmed death- ligand 1 expression, and pembrolizumab monotherapy was initiated. The patient exhibited fatigue from the 12th course(36 weeks after treatment initiation) of treatment. Chest computed tomography revealed scattered ground-glass opacities in the upper lobes of both lungs, and he was subsequently diagnosed with interstitial pneumonia. Fatigue persisted even after a drug holiday from pembrolizumab, and the patient was diagnosed with hypopituitarism based on the results of endocrinological examinations. Rashes appeared on both legs 40 weeks after treatment initiation, which led to the patient being diagnosed with a drug-induced skin disorder. All the adverse events resolved upon treatment with hydrocortisone. Immune- related adverse events due to pembrolizumab may occur in multiple organs simultaneously.

The Multi-Domain Intervention Trial in Older Adults With Diabetes Mellitus for Prevention of Dementia in Japan: Study Protocol for a Multi-Center, Randomized, 18-Month Controlled Trial.

Background: The Japan-Multi-domain Intervention Trial for Prevention of Dementia in Older Adults with Diabetes (J-MIND-Diabetes) is an 18-month, multi-centered, open-labeled, randomized controlled trial designed to identify whether multi-domain intervention targeting modifiable risk factors for dementia could prevent the progression of cognitive decline among older adults with type 2 diabetes mellitus (T2DM). This manuscript describes the study protocol for the J-MIND-Diabetes trial. Materials and Methods: Subjects of this trial will comprise a total of 300 T2DM outpatients aged 70-85 years with mild cognitive impairment. Subjects will be centrally randomized into intervention and control groups at a 1:1 allocation ratio using the stratified permuted-block randomization methods. The intervention group will participate in multi-domain intervention programs aimed at: (1) management of metabolic and vascular risk factors; (2) physical exercise and self-monitoring of physical activity; (3) nutritional guidance; and (4) social participation. The control group will receive usual T2DM care and general instructions on dementia prevention. The primary and secondary outcomes will be assessed at baseline, at 6- and 18-month follow-up. The primary outcome is change from baseline at 18 months in a global composite score combining several neuropsychological domains, including global cognitive function, memory, attention, executive function, processing speed and language. Secondary outcomes include: (1) cognitive changes in neuropsychological tests; (2) changes in geriatrics assessments; (3) metabolic control and diabetic complications; (4) changes in blood and urinary markers. Discussion: This trial will be the first trial to demonstrate the effectiveness of multi-domain intervention in preventing cognitive decline in older adults with T2DM at increased risk of dementia in Japan. Trial Registration: UMIN000035911; Registered on the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) 18 February 2019. (https://upload.umin.ac.jp/cgi-open-bin/ctr_e/ctr_view.cgi?recptno=R000040908).

Analysis of time-dependent alterations of parameters related to erythrocytes after ipragliflozin initiation.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors often increase the hematocrit. It remains unclear whether this increase would be observed in all patients administered SGLT2 inhibitors, however. We therefore used the data from the previous study and investigated time-dependent alterations of various outcomes related to erythrocytes, erythropoiesis, and clinical outcome in type 2 diabetes subjects (n = 89) treated with ipragliflozin for 16 weeks. Among a total of 89 participants, 71 subjects (80.0% of total participants) showed the elevation of the hematocrit and 18 subjects (20.0% of total participants) did not at 16 weeks. Although the hematocrit levels at baseline were significantly lower in hematocrit-elevated group than non-elevated group, they reached the same levels 4 weeks after the onset of treatment. Binomial logistic regression analysis demonstrated that a lower baseline hematocrit level was related to the elevation of hematocrit at 16 weeks. Optimal cutoff hematocrit levels at baseline to predict hematocrit elevation were 46.9% (male) and 41.7% (female) in ROC analysis. Random intercept model analysis revealed the serum erythropoietin level increased in both hematocrit-elevated and non-elevated groups, whereas only the former group showed an increase in the percentage of reticulocytes during the first 4 weeks. These results suggest that the ipragliflozin-induced increase in hematocrit which is affected by the baseline hematocrit level is attributable to the responsiveness to, but not to the production of, erythropoietin. Collectively, Ht elevation observed in administration of SGLT2 inhibitors can result from erythropoietin-induced erythropoiesis, which is determined by the pre-treatment Ht level. Trial registration: This trial has been registered with University Hospital Medical Information Network Clinical Trial Registry (UMIN-CTR no. 000015478).

Relationship between glycated hemoglobin level and duration of hypoglycemia in type 2 diabetes patients treated with sulfonylureas: A multicenter cross-sectional study.

AIMS/INTRODUCTION: Sulfonylurea-related hypoglycemia increases the risk of cardiovascular sequela, such as cardiac arrhythmia. This study aimed to clarify the relationship between the level of glycated hemoglobin (HbA1c ) and the duration of hypoglycemia in type 2 diabetes patients treated with sulfonylureas. MATERIALS AND METHODS: Glucose levels in the enrolled patients (n = 300) were investigated with a professional continuous glucose monitoring device in the outpatient setting at six diabetes centers in Japan. RESULTS: A total of 269 participants completed the study. The duration of hypoglycemia with glucose values of <54 mg/dL was significantly longer in patients with an HbA1c level of ≤6.4% than in those with an HbA1c level of ≥8.0%, and that of hypoglycemia with glucose values of <70 mg/dL was significantly longer in patients with an HbA1c level of ≤6.4%, 6.5-6.9% or 7.0-7.4% than in those with an HbA1c level of ≥8.0%. Patients with an HbA1c level of ≤6.4% were exposed to glucose values of <70 mg/dL for >10% of the time in daily life (6.8 ± 5.6 min/h). The duration of hypoglycemia with glucose values of <70 mg/dL was longer at night than during the daytime, and the nadir of glucose values occurred between 03.00 and 05.00 hours irrespective of HbA1c level. The duration of hypoglycemia was associated with the duration of diabetes and sulfonylurea dose. CONCLUSIONS: The duration of hypoglycemia was inversely correlated with HbA1c level and was longer during the night-time than daytime in type 2 diabetes patients treated with sulfonylureas.

Effects of ipragliflozin on glycemic control, appetite and its related hormones: A prospective, multicenter, open-label study (SOAR-KOBE Study).

AIMS/INTRODUCTION: Sodium-glucose cotransporter 2 (SGLT-2) inhibitors improve blood glucose control, as well as reducing bodyweight by promoting urinary glucose excretion. The weight loss is less than expected from urinary glucose loss, however, likely because of an increase in food intake. To investigate whether SGLT-2 inhibitors might increase appetite by affecting related hormones, we examined the effects of the SGLT-2 inhibitor, ipragliflozin, including those on appetite-regulating hormones, in individuals with suboptimally controlled type 2 diabetes. MATERIALS AND METHODS: The present prospective, multicenter, open-label study was carried out with 96 patients with a body mass index of ≥22 kg/m2 who were treated with ipragliflozin (50 mg/day) for 16 weeks. Parameters including glycated hemoglobin level, bodyweight, circulating leptin and active ghrelin concentrations, and appetite as assessed with a visual analog scale were measured before and during treatment. RESULTS: Both glycated hemoglobin level (from 7.9 ± 0.8 to 7.1 ± 0.7%) and bodyweight (from 75.2 ± 12.6 to 72.6 ± 12.4 kg) were significantly decreased after treatment for 16 weeks. The fasting serum leptin level was significantly decreased after 2 weeks (from 19.5 ± 13.1 to 18.1 ± 12.4 ng/mL) and remained decreased up to 16 weeks, even after adjustment for bodyweight, whereas the plasma active ghrelin level showed no significant change. The visual analog scale score for hunger was significantly increased at 2 and 8 weeks. CONCLUSIONS: The present results suggest that ipragliflozin improved glycemic control and reduced bodyweight, but also reduced serum leptin levels and might thereby have increased appetite.

Effects of Insulin Degludec and Insulin Glargine U300 on Day-to-Day Fasting Plasma Glucose Variability in Individuals with Type 1 Diabetes: A Multicenter, Randomized, Crossover Study (Kobe Best Basal Insulin Study 2).

INTRODUCTION: Administered basal insulin markedly influences the fasting plasma glucose (FPG) level of individuals with type 1 diabetes. Insulin degludec (IDeg) and insulin glargine U300 (IGlar U300) are now available as ultra-long-acting insulin formulations, but whether or how their glucose-stabilizing effects differ remains unclear. We will compare the effects of these basal insulins on parameters related to blood glucose control, with a focus on day-to-day glycemic variability, in individuals with type 1 diabetes treated with multiple daily injections. METHODS: A multicenter, randomized, open-label, crossover, comparative study (Kobe Best Basal Insulin Study 2) will be performed at 13 participating institutions in Japan. A total of 46 C-peptide-negative adult outpatients with type 1 diabetes will be randomly assigned 1:1 by a centralized allocation process to IGlar U300 (first period)/IDeg (second period) or IDeg (first period)/IGlar U300 (second period) groups, in which subjects will be treated with the corresponding basal insulin for consecutive 4-week periods. The basal insulin will be titrated to achieve an FPG of less than 130 mg/dL initially and then less than 110 mg/dL if feasible. In the last week of each period, plasma glucose will be determined seven times a day by self-monitoring of blood glucose (SMBG) and intraday and day-to-day glucose excursions will be determined by flash glucose monitoring (FGM). The primary end point is comparison of day-to-day glycemic variability as evaluated by the standard deviation (SD) of FPG during the last week of each treatment period. Secondary end points include the coefficient of variance of FPG, the frequency of severe hypoglycemia as evaluated by SMBG, the duration of hypoglycemia as evaluated by FGM, intraday glycemic variability calculated from both SMBG and FGM data, and the administered insulin dose. PLANNED OUTCOMES: The results of the study will be submitted for publication in a peer-reviewed journal to report differences in the effects of two ultra-long-acting basal insulins, IDeg and IGlar U300. CONCLUSION: This head-to-head comparison will be the first study to compare the effects of IDeg and IGlar U300 on day-to-day FPG variability in C-peptide-negative individuals with type 1 diabetes. TRIAL REGISTRATION: Registered in University Hospital Medical Information Network (UMIN) Clinical Trials Registry as 000029630 on 20 June 2017. FUNDING: Novo Nordisk Pharma Ltd.

Comprehensive Evaluation of Combination Therapy with Basal Insulin and Either Lixisenatide or Vildagliptin in Japanese Patients with Type 2 Diabetes: A Randomized, Open-Label, Parallel-Group, Multicenter Study.

INTRODUCTION: We comprehensively evaluated the effects of combination therapy with insulin glargine and the incretin-based drugs lixisenatide or vildagliptin in Japanese patients with type 2 diabetes. METHODS: In this 12-week, randomized, open-label, parallel-group, multicenter study (GLP-ONE Kobe), the incretin-based drug sitagliptin was randomly switched to lixisenatide (20 µg/day, n = 18) or vildagliptin (100 mg/day, n = 20) in patients with inadequate glycemic control despite combination therapy with insulin glargine and sitagliptin. The dose of insulin glargine was titrated after the switch to maintain fasting blood glucose at approximately 110 mg/dL. The primary end points of the study were the change in glycosylated hemoglobin (HbA1c) level between before and 12 weeks after the treatment switch, the proportion of patients achieving an HbA1c level below 7.0%, and the postprandial increase in glucose concentration as assessed by self-monitoring of blood glucose. RESULTS: The change in HbA1c level from baseline to 12 weeks did not differ significantly between the lixisenatide and vildagliptin groups (- 0.6 ± 0.7% and - 0.6 ± 1.2%, respectively, P = 0.920). Neither the proportion of patients achieving an HbA1c level below 7.0% nor the postprandial increase in glucose concentration was different between two groups. Body weight and serum low density lipoprotein (LDL) cholesterol level decreased significantly in the lixisenatide and vildagliptin groups, respectively. Both drugs were associated with mild gastrointestinal symptoms but not with severe hypoglycemia. Vildagliptin was associated with elevation of serum aspartate transaminase. Treatment satisfaction as assessed with the Diabetes Treatment Satisfaction Questionnaire did not differ significantly between the two groups. CONCLUSION: The combinations of basal insulin and either lixisenatide or vildagliptin have similar efficacies with regard to improvement of glycemic control. TRIAL REGISTRATION: This trial has been registered with UMIN (No. 000010769).

Very low-dose lenalidomide therapy for elderly multiple myeloma patients.

Lenalidomide treatment for refractory or relapsed multiple myeloma in elderly patients may be feasible in an outpatient setting. However, difficulties have been associated with the management of adverse effects. Therefore, a dose reduction in lenalidomide has been recommended in some cases. In this report, we encountered the successful treatment of myeloma in 6 elderly patients (aged above 70 years) with very low-dose lenalidomide (5 mg daily). Four patients exhibited more than a partial response with an 8.6 months median follow-up period, which was comparable with previous findings. The major adverse effect observed was infection, which occurred during the first several cycles. Others were less toxic, especially the absence of grade 3/4 toxicities for hematological adverse effects.Although a dose reduction in lenalidomide therapy for elderly patients is controversial, a very low dose could be safe and effective. Our group is currently conducting a multi-center prospective trial to evaluate the efficacy of low-dose lenalidomide therapy.

The distribution of several elements in cat urine and the relation between the content of elements and urolithiasis.

The concentrations of elements in urine obtained from cats with urolithiasis were compared with those of healthy cats. The concentration of several elements, such as sodium (Na), phosphorus (P), sulfur (S), and potassium (K), in urine obtained from cats with urolithiasis was significantly higher than that of healthy cats. A significant correlation (p<0.01) was found between the concentration of magnesium (Mg) and that of other elements, such as P (r=0.8913), S (r=0.6817), and K (r=0.8391), in the urine obtained from healthy cats. A significant correlation (r=0.7422, p<0.05) was also obtained between the concentration of K and that of P in urine collected from cats with urolithiasis, but the slope of regression line was significantly different from that of the urine obtained from healthy cats. Other correlations observed in healthy cats were not obtained from cats with urolithiasis. However, a significant correlation between the concentration of magnesium (Mg) and that of calcium was obtained only from cats with urolithiasis. The results of the present study suggest that urinary concentrations of various elements in cats with urolithiasis are higher than those of healthy cats. Furthermore, the balance of elements in the urine of cats with urolithiasis was altered.

Administration of a determinant of preproinsulin can induce regulatory T cells and suppress anti-islet autoimmunity in NOD mice.

Antigen-specific immunotherapy is expected to be an ideal strategy for treating type 1 diabetes (T1D). We investigated the therapeutic efficacy of a peptide in the leader sequence of preproinsulin, which was selected because of its binding affinity to the MHC I-A(g7) molecule. Preproinsulin-1 L7-24 peptide (L7-24) emulsified in Freund's incomplete adjuvant was administered subcutaneously to NOD mice. Administration of L7-24 increased the proportion of regulatory T cells in the spleen. Splenocytes of NOD mice immunized with this peptide secreted IL-4 and IL-10 in response to L7-24. This peptide also significantly prevented the development of diabetes and cured some newly diabetic NOD mice without recurrence. L7-24 peptide, which has a high affinity for pockets of I-A(g7), induced regulatory T cells and showed therapeutic effects. This peptide may provide a new approach for developing antigen-specific immunotherapy for autoimmune diabetes.

Regulatory CD8+ T cells induced by exposure to all-trans retinoic acid and TGF-beta suppress autoimmune diabetes.

Antigen-specific regulatory CD4(+) T cells have been described but there are few reports on regulatory CD8(+) T cells. We generated islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific regulatory CD8(+) T cells from 8.3-NOD transgenic mice. CD8(+) T cells from 8.3-NOD splenocytes were cultured with IGRP, splenic dendritic cells (SpDCs), TGF-beta, and all-trans retinoic acid (ATRA) for 5days. CD8(+) T cells cultured with either IGRP alone or IGRP and SpDCs in the absence of TGF-beta and ATRA had low Foxp3(+) expression (1.7+/-0.9% and 3.2+/-4.5%, respectively). In contrast, CD8(+) T cells induced by exposure to IGRP, SpDCs, TGF-beta, and ATRA showed the highest expression of Foxp3(+) in IGRP-reactive CD8(+) T cells (36.1+/-10.6%), which was approximately 40-fold increase compared with that before induction culture. CD25 expression on CD8(+) T cells cultured with IGRP, SpDCs, TGF-beta, and ATRA was only 7.42%, whereas CD103 expression was greater than 90%. These CD8(+) T cells suppressed the proliferation of diabetogenic CD8(+) T cells from 8.3-NOD splenocytes in vitro and completely prevented diabetes onset in NOD-scid mice in cotransfer experiments with diabetogenic splenocytes from NOD mice in vivo. Here we show that exposure to ATRA and TGF-beta induces CD8(+)Foxp3(+) T cells ex vivo, which suppress diabetogenic T cells in vitro and in vivo.

NO-mediated cytotoxicity contributes to multiple low-dose streptozotocin-induced diabetes but not to NOD diabetes.

Type 1 diabetes (T1D) is caused mostly by autoimmune destruction of pancreatic beta-cells, the precise mechanism of which remains unclear. Two major effector mechanisms have been proposed: direct cell-mediated and indirect cytokine-mediated cytotoxicity. Cytokine-mediated beta-cell destruction is presumed mainly caused by NO production. To evaluate the role of iNOS expression in T1D, this study used a novel iNOS inhibitor ONO-1714. ONO-1714 significantly reduced cytokine-mediated cytotoxicity and NO production in both MIN6N9a cells and C57BL/6 islets in the presence of IL-1beta, TNF-alpha, and IFN-gamma. To evaluate whether NO contributes to diabetes progression in vivo, ONO-1714 was administered to four different mouse models of autoimmune diabetes: multiple low-dose STZ (MLDS)-induced C57BL/6, CY-induced, adoptive transfer and spontaneous NOD diabetes. Exposure to STZ in vitro induced NO production in MIN6N9a cells and C57BL/6 islets, and in vivo injection of ONO-1714 to MLDS-treated mice significantly reduced hyperglycemia and interestingly, led to complete suppression of cellular infiltration of pancreatic islets. In contrast, when ONO-1714 was injected into spontaneous NOD mice and CY-induced and adoptive transfer models of NOD diabetes, overt diabetes could not be inhibited in these models. These findings suggest that NO-mediated cytotoxicity significantly contributes to MLDS-induced diabetes but not to NOD diabetes.

One amino acid difference is critical for suppression of the development of experimental autoimmune diabetes (EAD) with intravenous injection of insulinB:9-23 peptide.

InsulinB:9-23 peptide (insB:9-23) reactive T cells has been reported as crucial for type 1 diabetes. In this study, experimental autoimmune diabetes (EAD) mice, which subcutaneous immunization of ins1 or 2B:9-23 induced autoimmune diabetes in F1(B7.1B6 x BALB/c), was investigated for antigen specific therapy to delete pathogenic T cells. Intravenous injection of ins1 or 2B:9-23 significantly delayed the development of diabetes on the corresponding peptide-induced EAD (ins1EAD or ins2EAD) concomitant with reduced insulitis and insulin autoantibodies expression. Population of Foxp3(+) CD4(+) T cell was unchanged whereas the level of anti-insB:9-23 specific IgG(2a) but not IgG(1) were specifically decreased, suggesting reduction of pathogenic insB:9-23 reactive T cells. Most interestingly, intravenous administration of ins2B:9-23, whose amino acid sequence had one amino acid difference at position 9 delayed the development of diabetes in both ins1EAD and ins2EAD whereas ins1B:9-23 administration delayed diabetes in the ins1EAD but not ins2EAD, suggesting that one amino acid difference gives critical influence on the effect of intravenous injection of antigenic peptide for type 1 diabetes.

Prevention of recurrent but not spontaneous autoimmune diabetes by transplanted NOD islets adenovirally transduced with immunomodulating molecules.

Pancreatic islet transplantation has the potential to maintain normoglycemia in patients with established type 1 diabetes, thereby obviating the need for frequent insulin injections. Our previous study showed that recombinant IL-12p40-producing islets prevented the recurrence of NOD diabetes. First, to see which immunomodulating molecule-secreting islet grafts can most powerfully prevent diabetes development in NOD mice without immunosuppressant, NOD islets were transfected with one of the following adenoviral vectors: Ad.IL-12p40, Ad.TGF-beta, Ad.CTLA4-Ig, or Ad.TNF-alpha after which they were transplanted under the renal capsule of acutely diabetic NOD mice. The immunomodulating molecules produced by these adenovirus-transfected islets in vitro were 74+/-19ng, 50+/-4ng, 821+/-31ng, and 77+/-18ng/100 islets, respectively. Transplantation of IL-12p40, TNF-alpha, and CTLA4-Ig but not TGF-beta-secreting islets displayed enhanced survival and delayed diabetes recurrence in recent-onset diabetic recipients. IL-12p40-producing islet grafts most powerfully prevented recurrent diabetes in NOD mice. In addition, local production of TNF-alpha and CTLA4-Ig significantly prolonged islet graft survival. In second series of experiment, these manipulated islets were transplanted under the renal capsule of 10-week-old NOD recipients and were also transplanted subcutaneously into 2-week-old NOD recipients. Transplantation of these islets into 2- or 10-week-old pre-diabetic mice failed to protect them from developing diabetes; in fact, transplantation of Ad.TNF-alpha-transfected islets into 2-week-old mice actually accelerated diabetes onset. Taken together, this approach was ineffectual as a prophylactic protocol. In conclusion, this study showed comparisons of the immunomodulating effects of 4 different adenoviral vectors in the same transplantation model and local production of IL-12p40, TNF-alpha and CTLA4-Ig significantly prevented recurrent diabetes in NOD mice.

Intravenous administration of proinsulin 1 or 2-expressing fiber-mutant recombinant adenovirus vector protects against the development of diabetes in NOD mice.

Insulin has been reported as a major autoantigen in both human and murine type 1 diabetes (T1D). Insulin1-knockout NOD mice with only insulin2 are protected against the development of autoimmune diabetes, suggesting that insulin1 has strong immunogenicity and insulin2 has weak immunogenicity or a possible protective role in the pathogenesis of type 1 diabetes. In this study, we have developed fiber-mutant adenovirus vectors that express murine proinsulin1 or proinsulin2 (named Ad.Pins1-RGD/Ad.Pins2-RGD) and administered those virus vectors to the NOD mouse to evaluate modulation of autoimmune responses. The intravenous administration of either Ad.Pins1-RGD or Ad.Pins2-RGD at 3 and 5 weeks of age strongly suppressed the development of overt diabetes, accompanied by a significant reduction of insulin autoantibody (IAA), and suppression of disease was similar between administration of Ad.Pins1-RGD and that of Ad.Pins2-RGD. Our study suggests that systemic administration of fiber-mutant adenovirus vectors, which induce transient expression of proinsulin, may be applicable to a gene therapy inducing tolerance to insulin.

Autoreactive T cell response in CD25-negative fraction of peripheral blood mononuclear cells in established type 1 diabetes.

CD4(+)CD25(+) T cells (Tregs) play a critical role in maintaining dominant peripheral tolerance, and pathogenic autoreactive T cells may be frequent in the CD25-negative fraction of peripheral blood mononuclear cells (PBMCs) from patients with autoimmune disease. We therefore investigated whether T cell autoimmune responses to recombinant GAD65 can be detected by the use of ELISPOT assay in the CD25-negative fraction of PMBCs from Japanese type 1 diabetes (T1D) patients. The frequency of CD4(+)CD25(+) T cells was not different among patients with newly developed T1D, established T1D, and healthy controls. The CD25 positive cell-depleted fraction was obtained by negative selection with antihuman CD25 magnetic beads, reducing the number of CD4(+)CD25(+) T cells from 4-5% to less than 1%. In whole PBMC fraction, there was a significant elevation of IFN-gamma spots in PBMCs from recently diagnosed patients with T1D (P < 0.05), whereas the number of IFN-gamma spots from patients with established T1D was not significant. In the CD25-negative fraction, unlike whole PBMCs, we observed the significant IFN-gamma spots to GAD65 in the fraction from patients with established T1D (P < 0.05), but not in those with recently diagnosed disease. The phenomena were not observed for IL-4 spots. Our data suggest a possible role of Tregs maintaining dominant peripheral tolerance in T1D and application of further improved T cell assay detecting autoimmunity even in established T1D.

Immunological aspects of 'fulminant type 1 diabetes'.

'Fulminant diabetes' has been recognized as a super-acute onset and non-autoimmune type 1 diabetes. To evaluate autoimmunity against pancreatic beta cell in fulminant diabetes, ELISPOT assay was applied to the peripheral blood of these patients. In our ELISPOT system, GAD65-reactive and insulin B9-23-reactive IFN-gamma spots were detected in 46.3 and 26.0% of autoantibody-positive type 1 diabetes. Also, in fulminant type 1 diabetic patients, IFN-gamma spots in response to GAD65 and insulin B9-23 peptide were detected in 69.2 and 25.0%, respectively. These results suggest that anti-beta cell autoimmunity contributes to develop fulminant type 1 diabetes. Fulminant type 1 diabetes is known to have IDDM-resistant HLA DR2 with similar frequency of non-T1D subjects. In a mouse model, when islet-reactive CD8 cells are transferred to young NOD mice, the recipients develop overt diabetes within 1 week with massive insulitis. In (NOD x Balb/c) F1 mice, which hold idd-resistant genes, transfer of islet-reactive CD8 cells induced diabetes to 60% F1 recipients within 1 week with the later disappearance of insulitis. This mouse model shows very similar feathers to fulminant type 1 diabetes; idd-resistant HLA and no insulitis. These results implicated that once anti-islet immunity is optimally activated, subjects with partially resistant alleles could become overt diabetes.