Efficacy and safety of liraglutide monotherapy compared with metformin in Japanese overweight/obese patients with type 2 diabetes.

There is little information on direct comparison between metformin and glucagon-like peptide-1 (GLP-1) receptor agonists in the Asian population. This study examined the efficacy and safety of liraglutide monotherapy compared with metformin monotherapy in overweight/obese Japanese patients with type 2 diabetes (T2DM). The study was a 24-week, open-labeled, randomized controlled study. Overweight or obese patients with T2DM aged 20-75 years with suboptimal glycemic control were randomized to liraglutide or metformin monotherapy. The primary endpoint was change in HbA1c at week 24. Secondary endpoints included changes in daily glycemic profile, body weight, incidence of hypoglycemia and other adverse events. The study, which was originally planned to enroll 50 subjects in each group, was ended with insufficient recruitment. A total of 46 subjects completed the study, and analysis was conducted in this cohort. Reduction in HbA1c at week 24 was comparable between the metformin (n = 24) and liraglutide (n = 22) groups (-0.95 ± 0.80% vs. -0.80 ± 0.88%, p = 0.77), while the liraglutide group reached maximal reduction more rapidly than did the metformin group. There was no significant difference in weight gain or incidence of hypoglycemia between the groups. Diarrhea was more frequent in the metformin group, while constipation was more frequent in the liraglutide group. There was no significant difference in treatment satisfaction between the groups. In conclusion, liraglutide and metformin monotherapy showed similar reduction in HbA1c during 24 weeks, with no difference in weight gain or incidence of hypoglycemia in overweight or obese Japanese patients with T2DM.

Pioglitazone may accelerate disease course of slowly progressive type 1 diabetes.

BACKGROUND: It has been reported that intervention with insulin in slowly progressive type 1 diabetic (SPIDDM) patients delays the progression to an insulin-dependent state compared to that with sulfonylureas. However, the rate of progression to SPIDDM with the use of insulin-sensitizing agents is unknown. The aim of this study was to determine the effect of insulin-sensitizing agents on SPIDDM patients. METHODS: The enrolled SPIDDM patients were randomly allocated to a pioglitazone or metformin group. When the haemoglobin A1C level was more than 8% on two consecutive occasions, the case was considered to reach the end point. RESULTS: By 4 years post-intervention, all patients had reached the end point in the pioglitazone group, whereas only 20% of patients had reached the end point in the metformin group (p<0.05). CONCLUSIONS: Pioglitazone may accelerate the disease course of SPIDDM.

CXC chemokine ligand 10 DNA vaccination plus Complete Freund's Adjuvant reverses hyperglycemia in non-obese diabetic mice.

OBJECTIVE: Complete Freund's Adjuvant (CFA) is known to arrest autoimmune diabetes development in non-obese diabetic (NOD) mice. However, CFA alone cannot induce effective remission in diabetic NOD mice. Previously, we reported that anti-CXC chemokine ligand 10 (CXCL10) antibody can promote beta-cell proliferation in NOD mice. In the present study, we aimed to examine whether anti-CXCL10 plus CFA treatment can effectively reverse autoimmune diabetes development. METHODS: Systemic supply of anti-CXCL10 antibody by CXCL10 DNA vaccination in combination with CFA injection was performed in new-onset diabetic NOD mice. Remission rate of diabetes, histological characteristics of residual insulitis lesions, residual beta-cell mass, and regulatory T cell population in local pancreas were examined. RESULTS: A high frequency of diabetes reversal was observed after combination treatment with anti-CXCL10 plus CFA. In mice showing diabetes reversal, residual beta-cell mass was significantly increased, and some beta-cells were in a proliferative state. Although systemic cytokine profiles were unaffected, the frequency of "hybrid regulatory T cells", i.e. regulatory T cells expressing CXCR3, was significantly increased in local pancreatic lesions. This was possibly associated with the regulation of anti-islet autoimmunity. CONCLUSIONS: Anti-CXCL10 plus appropriate immune adjuvant therapy arrested, and reversed, type 1 diabetes development.

Increased risk of renal deterioration associated with low e-GFR in type 2 diabetes mellitus only in albuminuric subjects.

OBJECTIVE: The significance of estimated glomerular filtration rate (e-GFR) in diabetic nephropathy has yet to be clearly determined. We therefore compared albuminuria and e-GFR for usefulness in predicting progressive decline in renal function. METHODS: A total of 1,303 subjects with type 2 diabetes mellitus whose e-GFR was more than 30 mL/min/1.73 m(2) were followed for three years. Associations of clinical staging based on AER and that based on e-GFR with progression of renal insufficiency (e-GFR <30 mL/min/1.73 m(2)) were evaluated. RESULTS: On univariate analysis, both clinical stages based on e-GFR and AER were significant variables (p<0.05). On multiple logistic regression analysis, the odds ratio for macroalbuminuria was 132.3, and that for microalbuminuria was 10.3 while that for e-GFR less than 60 mL/min/1.73 m(2) was 9.0 for further deterioration of renal function. On the other hand, subjects without albuminuria exhibited a rate of disease progression of less than 1% irrespective of e-GFR level. CONCLUSIONS: Both albuminuria and reduced e-GFR are significant and independent risk factors for further deterioration of diabetic nephropathy, though albuminuria had a greater odds ratio than reduced e-GFR for deterioration of renal function over a three-year period. e-GFR exhibited additive risk for deterioration of diabetic nephropathy within three years only when albuminuria was present.

Hyperplastic islets observed in "reversed" NOD mice treated without hematopoietic cells.

At the onset of type 1 diabetes, most of the insulin-producing pancreatic beta cells are destroyed by effector cells, and therefore, the following two factors, at a minimum, are necessary for "reversing" hyperglycemia in autoimmune diabetes; depletion of effector cells and enhancement of beta cell regeneration. In this study, we tried a novel approach for "reversing" autoimmune diabetes in a murine model. Here we show that remission could be achieved with a combination therapy of a single injection of complete Freund's adjuvant (CFA) and a single intraperitoneal injection of a pancreatic beta cell line, MIN6N-9a, in recent-onset diabetic NOD (non-obese diabetic) mice. Five out of seven mice (71%) receiving MIN6N-9a and CFA became normoglycemic within 120 days after treatment, whereas only two of nine (22%) receiving vehicle instead of MIN6N-9a achieved remission. Histological examination of pancreatic specimens from "reversed" mice showed decreased islet number, but each islet was markedly hyperplastic; being about six times larger than those from controls. Although it has been reported that hematopoietic cells such as splenocytes differentiate into insulin-producing cells and play a key role, our data indicate that they are not an absolute requirement for the "reversal" of autoimmune diabetes.

Human chorionic gonadotropin prevents Sjögren's syndrome-like exocrinopathy in mice.

OBJECTIVE: Results of recent studies suggest that human chorionic gonadotropin (HCG), a placental glycoprotein hormone required for the maintenance of pregnancy, may have immunomodulatory properties. Primary Sjögren's syndrome (SS), a chronic autoimmune disorder of unknown etiology, affects multiple exocrine glands including the salivary and lacrimal glands. The purpose of the present study was to determine whether HCG could prevent the development of salivary gland exocrinopathy in NOD mice, an experimental model of Sjögren's-like syndrome. METHODS: Female NOD mice were treated with HCG from 6 weeks of age to 12 weeks of age. At 14 weeks, tissue samples were evaluated for inflammatory lesions and cytokine messenger RNA by real-time polymerase chain reaction. At 18 weeks, the salivary flow rate was measured. RESULTS: Treatment with HCG resulted in a significant decrease in lymphocyte infiltration and parenchymal cell damage in the submandibular salivary glands. Messenger RNA levels of interferon-gamma, tumor necrosis factor alpha, interleukin-1beta, and interleukin-10, as well as inducible nitric oxide synthase and matrix metalloproteinase 9, were significantly decreased. Function studies revealed a marked increase in the salivary flow rate in HCG-treated mice compared with that in phosphate buffered saline-treated mice. CONCLUSION: In NOD mice, HCG acts as an immune modulator and prevents the development of salivary gland exocrinopathy. These findings suggest that HCG, a naturally occurring reproductive hormone, may be useful in the treatment of Sjögren's syndrome and other human autoimmune diseases.

Glucagon-like peptide-1 gene therapy in obese diabetic mice results in long-term cure of diabetes by improving insulin sensitivity and reducing hepatic gluconeogenesis.

Long-term treatment with glucagon-like peptide (GLP)-1 or its analog can improve insulin sensitivity. However, continuous administration is required due to its short half-life. We hypothesized that continuous production of therapeutic levels of GLP-1 in vivo by a gene therapy strategy may remit hyperglycemia and maintain prolonged normoglycemia. We produced a recombinant adenovirus expressing GLP-1 (rAd-GLP-1) under the cytomegalovirus promoter, intravenously injected it into diabetic ob/ob mice, and investigated the effect of this treatment on remission of diabetes, as well as the mechanisms involved. rAd-GLP-1-treated diabetic ob/ob mice became normoglycemic 4 days after treatment, remained normoglycemic over 60 days, and had reduced body weight gain. Glucose tolerance tests found that exogenous glucose was cleared normally. rAd-GLP-1-treated diabetic ob/ob mice showed improved beta-cell function, evidenced by glucose-responsive insulin release, and increased insulin sensitivity, evidenced by improved insulin tolerance and increased insulin-stimulated glucose uptake in adipocytes. rAd-GLP-1 treatment increased basal levels of insulin receptor substrate (IRS)-1 in the liver and activation of IRS-1 and protein kinase C by insulin in liver and muscle; increased Akt activation was only observed in muscle. rAd-GLP-1 treatment reduced hepatic glucose production and hepatic expression of phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, and fatty acid synthase in ob/ob mice. Taken together, these results show that a single administration of rAd-GLP-1 results in the long-term remission of diabetes in ob/ob mice by improving insulin sensitivity through restoration of insulin signaling and reducing hepatic gluconeogenesis.

Prolonged Remission of Diabetes by Regeneration of ß Cells in Diabetic Mice Treated with Recombinant Adenoviral Vector Expressing Glucagon-like Peptide-1.

Type 1 diabetes results from insulin deficiency caused by destruction of pancreatic ß cells. Glucagon-like peptide (GLP)-1 stimulates ß cell growth and differentiation. To determine whether continuous expression of GLP-1 in vivo can regenerate ß cells and remit type 1 diabetes in mice for a prolonged time, we constructed an adenoviral vector containing the cytomegalovirus promoter/enhancer and albumin leader sequence followed by GLP-1 cDNA (rAd-GLP-1). A single administration of rAd-GLP-1 via the tail vein into streptozotocin (STZ)-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in remission of diabetes within 10 days; normoglycemia remained until the experiment was terminated. The number of insulin-positive cells in the pancreas and insulin secretion significantly increased in rAd-GLP-1-treated mice compared with STZ-induced diabetic mice treated with rAd-ß-galactosidase. Glucose tolerance tests in mice that achieved normoglycemia after treatment with rAd-GLP-1 showed that the kinetics of glucose clearance was similar to normal NOD/SCID mice. Treatment of autoimmune diabetic mice with rAd-GLP-1 restored normoglycemia, which was maintained for 1 year when mice were also treated with an immunoregulator to halt the autoimmune response to ß cells. We suggest that regeneration of insulin-producing cells by GLP-1 gene therapy may be a potential method for prolonged control of type 1 diabetes in humans.

Prolonged remission of diabetes by regeneration of beta cells in diabetic mice treated with recombinant adenoviral vector expressing glucagon-like peptide-1.

Type 1 diabetes results from insulin deficiency caused by destruction of pancreatic beta cells. Glucagon-like peptide (GLP)-1 stimulates beta cell growth and differentiation. To determine whether continuous expression of GLP-1 in vivo can regenerate beta cells and remit type 1 diabetes in mice for a prolonged time, we constructed an adenoviral vector containing the cytomegalovirus promoter/enhancer and albumin leader sequence followed by GLP-1 cDNA (rAd-GLP-1). A single administration of rAd-GLP-1 via the tail vein into streptozotocin (STZ)-induced diabetic non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in remission of diabetes within 10 days; normoglycemia remained until the experiment was terminated. The number of insulin-positive cells in the pancreas and insulin secretion significantly increased in rAd-GLP-1-treated mice compared with STZ-induced diabetic mice treated with rAd-beta-galactosidase. Glucose tolerance tests in mice that achieved normoglycemia after treatment with rAd-GLP-1 showed that the kinetics of glucose clearance was similar to normal NOD/SCID mice. Treatment of autoimmune diabetic mice with rAd-GLP-1 restored normoglycemia, which was maintained for 1 year when mice were also treated with an immunoregulator to halt the autoimmune response to beta cells. We suggest that regeneration of insulin-producing cells by GLP-1 gene therapy may be a potential method for prolonged control of type 1 diabetes in humans.

Frequency of CTLA-4 gene CT60 polymorphism may not be affected by vitamin D receptor gene Bsm I polymorphism or HLA DR9 in autoimmune-related type 1 diabetes in the Japanese.

One of the CTLA-4 SNPs, +6230G>A (CT60), has recently been reported to be related to susceptibility to type 1 diabetes and autoimmune thyroid disease. We have previously reported an association between acute-onset type 1 diabetes in Japanese and the Vitamin D receptor (VDR) gene Bsm I large B polymorphism, which is related to the Th1-type response. Moreover, we found a significant correlation between autoimmune-related type 1 diabetes with HLA DR9 and detection of GAD-reactive Th1 (T helper 1)-type cells. In the present article, we tried to clarify whether the frequency of one of the CTLA-4 SNPs, +6230G>A (CT60), is affected by the VDR gene Bsm I polymorphism or by HLA DR9 in Japanese type 1 diabetics. The frequency of the CT60 GG genotype did not appear to be affected by either the VDR gene Bsm I large B polymorphism or HLA DR9.

Thiazolidinediones may not reduce diabetes incidence in type 1 diabetes.

Thiazolidinediones improve glycemic control by reducing insulin resistance. Some studies have demonstrated that troglitazone had a preventative effect on diabetes in NOD (non-obese diabetic) mice. One of the mechanisms proposed for the prevention of diabetes by thiazolidinediones is an effect on T-helper 1/T-helper 2 (Th1/Th2) balance. In this article, we attempted to clarify whether pioglitazone is also effective in preventing diabetes as compared to metformin, which has no immunological effect. Female NOD mice were administered pioglitazone or metformin orally, and the insulitis score, cytokines secreted from splenocytes, cytokine expression levels in the pancreas, and the incidence of diabetes after acceleration by cyclophosphamide were analyzed. We could not find any advantage of pioglitazone in preventing Th1 skewing and the development of diabetes over metformin. Therefore, further research should take place before the application of thiazolidinediones to human slowly progressive insulin-dependent diabetes mellitus (SPIDDM) patients.

Significance of serum CXCL10/IP-10 level in type 1 diabetes.

Although we have previously reported an elevated serum level of CXCL10/IP-10 (CXCL10), a Th1 chemokine, in type 1 diabetic patients, little is known about the origin of serum CXCL10 and its significance in type 1 diabetes. Therefore, we examined serum CXCL10 level in NOD mice in association with the expression level of CXCL10 in the pancreas, pancreatic lymph nodes (LN) and spleen. Serum CXCL10 level increased over time towards the onset of diabetes, and was significantly higher in the "diabetic" period (20 and 24 weeks of age and at onset of diabetes) than in the "pre-diabetic" period (4, 8 and 16 weeks of age). Moreover, serum CXCL10 level was associated with CXCL10 and CXCR3 mRNA level in pancreatic LN. Furthermore, it seemed that serum CXCL10 level increased just before (or at) the onset of overt diabetes. These results suggest that serum CXCL10 level may reflect accumulation of Th1 lymphocytes in pancreatic LN, and measurement of serum CXCL10 level may be useful to assess the pathophysiology of the disease course in type 1 diabetes.

CXCL10 DNA vaccination prevents spontaneous diabetes through enhanced beta cell proliferation in NOD mice.

CXCL10, a chemokine for Th1 cells, is involved in the pathogenesis of various Th1-dominant autoimmune diseases. Type 1 diabetes is considered to be a Th1-dominant autoimmune disease, and a suppressive effect of CXCL10 neutralization on diabetes development has been reported in a cyclophosphamide-induced accelerated diabetes model through induction of beta cell proliferation. However, intervention in a diabetes model might bring about opposite effects, depending on the timing, amount, or method of treatment. In the present study, we examined the effect of CXCL10 neutralization in a "spontaneous diabetes" model of NOD mice, using CXCL10 DNA vaccination (pCAGGS-CXCL10). pCAGGS-CXCL10 treatment in young NOD mice induced the production of anti-CXCL10 Ab in vivo and suppressed the incidence of spontaneous diabetes, although this treatment did not inhibit insulitis or alter the immunological response. pCAGGS-CXCL10 treatment enhanced the proliferation of pancreatic beta cells, resulting in an increase of beta cell mass in this spontaneous diabetes model as well. Therefore, CXCL10 neutralization is suggested to be useful for maintaining beta cell mass at any stage of autoimmune diabetes.

Insulin-like growth factor-1 (IGF-1)-derived peptide protects against diabetes in NOD mice.

Spontaneous diabetes in non-obese diabetic (NOD) mice results from beta-cell destruction by autoreactive T lymphocytes. Here, we report the significance of insulin-like growth factor-1 (IGF-1) peptide as a tool for the prevention of type 1 diabetes. Female NOD mice were immunized with a subcutaneous injection of IGF-1, glutamic acid decarboxylase (GAD), insulin or IGF-1-derived peptides (residues 8-23, 24-41 or 50-70) in incomplete Freund's adjuvant (IFA) or with IFA only as the control group at 4 weeks of age, and observed up to 36-37 weeks of age. Diabetes onset was significantly suppressed and delayed in the IGF-1 group as compared to the GAD, insulin and control groups (p<0.05), and it was significantly suppressed and delayed in the (50-70)IGF-1 group as compared to the (8-23)IGF-1 and control groups (p<0.02). Although the degree of insulitis in all treated mice was not significantly different, a significant number of IL-4-producing cells in response to IGF-1 peptides were detected in (50-70)IGF-1-treated mice in intracellular cytokine assay. In conclusion, IGF-1 peptide 50-70 immunizations of NOD mice suppressed and delayed diabetes onset, probably through amplification of the Th2-type response. It was suggested that IGF-1 peptide 50-70 immunization can be used as a tool for prevention of type 1 diabetes.

CXC chemokine ligand 10 neutralization suppresses the occurrence of diabetes in nonobese diabetic mice through enhanced beta cell proliferation without affecting insulitis.

We have shown that neutralization of IFN-inducible protein 10/CXCL10, a chemokine for Th1 cells, breaks Th1 retention in the draining lymph nodes, resulting in exacerbation in Th1-dominant autoimmune disease models induced by immunization with external Ags. However, there have been no studies on the role of CXCL10 neutralization in Th1-dominant disease models induced by constitutive intrinsic self Ags. So, we have examined the effect of CXCL10 neutralization using a type 1 diabetes model initiated by developmentally regulated presentation of beta cell Ags. CXCL10 neutralization suppressed the occurrence of diabetes after administration with cyclophosphamide in NOD mice, although CXCL10 neutralization did not significantly inhibit insulitis and gave no influence on the trafficking of effector T cells into the islets. Because both CXCL10 and CXCR3 were, unexpectedly, coexpressed on insulin-producing cells, CXCL10 was considered to affect mature and premature beta cells in an autocrine and/or paracrine fashion. In fact, CXCL10 neutralization enhanced proliferative response of beta cells and resultantly increased beta cell mass without inhibiting insulitis. Thus, CXCL10 neutralization can be a new therapeutic target for beta cell survival, not only during the early stage of type 1 diabetes, but also after islet transplantation.

GAD-reactive T cells were mainly detected in autoimmune-related type 1 diabetic patients with HLA DR9.

Type 1 diabetes mellitus (T1DM) is considered to be a T cell-mediated disease, and many reports suggest that some HLA types, especially HLA DR4 and DR9, convey susceptibility to T1DM in Japanese. We investigated the association between T cell reactivity against GAD and HLA types in "islet-associated autoantibody-positive" T1DM in Japanese. Blood samples were obtained from 36 "autoantibody-positive" type 1 diabetic patients with HLA DR4 or DR9 and 23 type 2 diabetic patients with HLA DR4 or DR9 as controls. They were divided into three groups, DR4/9, DR4/X, and DR9/X groups. In each HLA type group, GAD-reactive IFN-gamma-producing CD4(+) cells were assessed by means of intracellular cytokine staining for flow cytometry. Type 1 diabetic patients with HLA DR9/X had significantly higher numbers of GAD-reactive IFN-gamma-producing CD4(+) cells as compared to type 1 diabetic patients with DR4/X or DR4/9 (P < 0.05) and all type 2 diabetic patients. There was no significant difference in the number of GAD-reactive IFN-gamma-producing CD4(+) cells between type 1 diabetic and type 2 diabetic patients belonging to the DR4/X and DR4/9 groups. There was an association between T cell reactivity against GAD and HLA DR9 in Japanese type 1 diabetes.

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase) P22 Phox C242T gene polymorphism in type 1 diabetes.

Type 1 diabetes is caused by the immune-mediated destruction of insulin-secreting pancreatic beta cells and is thought to be an autoimmune disease resulting from a complex interaction of genetic and environmental factors. In animal models of type 1 diabetes, macrophages and their products, superoxides, have central roles in the beta cell destruction, but in humans their roles remain unclear. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase produces superoxide in macrophages, and its essential component, p22 phox, is a critical enzyme for superoxide production. The C242T polymorphism in the p22 phox coding gene has been reported to be associated with reduced oxidase activity. We therefore investigated whether the p22 phox gene polymorphism affected the susceptibility to and clinical course of type 1 diabetes. We examined 287 Japanese type 1 diabetic patients and 425 unrelated nondiabetic subjects. In addition, we allocated the diabetic patients to the following three groups: (1) acute-onset type 1 diabetes with at least one autoantibody (GADA, IA-2, IAA); (2) acute-onset type 1 diabetes without autoantibodies; and (3) slow-onset type 1 diabetes with autoantibody. We could not find a significant difference in p22 phox genotype and T allele frequency between overall type 1 diabetic patients and control subjects. Regardless of the onset pattern and autoantibody positivity of type 1 diabetes, no difference in p22 phox genotype and T allele frequency was found among the groups. In conclusion, the p22 phox C242T gene polymorphism did not affect the susceptibility to and clinical course of Japanese type 1 diabetes.

Stromal cell-derived factor-1 chemokine gene polymorphism is not associated with onset age of Japanese type 1 diabetes.

Type 1 diabetes is characterized by cell-mediated autoimmune destruction of pancreatic beta cells. Although the disease shows a strong association with HLA class II alleles, other genes may influence the initiation or the rate of progression of the autoimmune process. Recently, it was reported that a polymorphism of the stromal cell-derived factor-1 (SDF-1) (a kind of chemokine) gene was associated with early onset of type 1 diabetes in Caucasians. Therefore, we examined SDF-1 gene polymorphism in Japanese type 1 diabetes in this study. We examined the SDF-1 gene polymorphism (801G-->A) in 298 unrelated Japanese type 1 diabetic patients and 270 healthy subjects by the TaqMan PCR method. Allelic and genotypic frequencies of the SDF-1 A variants were similar in overall type 1 diabetic patients and healthy subjects. We then stratified the patients by their onset pattern (acute vs. slow onset) and islet-associated autoantibody positivity. However, no significant difference was found among each group of type 1 diabetes. Furthermore, unlike the previous report in "Caucasian" type 1 diabetics, the SDF-1 A variant was not associated with early onset of the disease in Japanese type 1 diabetics. The SDF-1 gene polymorphism was not associated with onset age (or onset pattern) of type 1 diabetes in Japanese. Further study is necessary to conclude whether SDF-1 gene polymorphism affects the onset age in type 1 diabetes in general.

Two cases of "fulminant" type 1 diabetes suggesting involvement of autoimmunity.

Recently, a novel subtype of type 1 diabetes, so-called fulminant type 1 diabetes, has been proposed. One of the characteristics of this subtype is the absence of detectable "islet-associated" autoantibody, so it was originally proposed as being "nonautoimmune-mediated"; however, it has not yet been concluded whether autoimmunity is involved. We have previously shown that serum interferon-inducible protein-10 and glutamic acid decarboxylase-reactive CD4(+) interferon-gamma-producing cells in the peripheral blood are good markers for T cell-mediated autoimmunity in type 1 diabetes. Here, we report two cases of fulminant type 1 diabetes in which these markers were detected and in which the involvement of islet-associated autoimmunity is suggested.