Taro Maruyama

Systematic search for single nucleotide polymorphisms in a lymphoid tyrosine phosphatase gene (PTPN22): Association between a promoter polymorphism and type 1 diabetes in Asian populations

The protein tyrosine phosphatase, nonreceptor 22 gene (PTPN22) maps to human chromosome 1p13.3‐p13.1 and encodes an important negative regulator of T‐cell activation, lymphoid‐specific phosphatase (Lyp). Recently, the minor allele of a single‐nucleotide polymorphism (SNP) at nucleotide position 1858 (rs2476601, +1858C > T) was found to be associated with type 1 diabetes. However, the degree of the association is variable among ethnic populations, suggesting the presence of other disease‐associated variants in PTPN22. To examine this possibility, we carried out a systemic search for PTPN22 using direct sequencing of PCR‐amplified products in the Japanese population. Association and linkage studies were also conducted in 1,690 Japanese samples, 180 Korean samples, and 472 Caucasian samples from 95 nuclear families. We identified five novel SNPs, but not the +1858C > T SNP. Of these two frequent SNPs, −1123G > C, and +2740C > T were in strong linkage disequilibrium (LD), and the −1123G > C promoter SNP was associated with acute‐onset but not slow‐onset type 1 diabetes in the Japanese population (odds ratio [OR] = 1.42, 95% CI = 1.07–1.89, P = 0.015). This association was observed also in Korean patients with type 1 diabetes (Mantel–Haenszel χ2 = 6.543, P = 0.0105, combined OR = 1.41 95% CI = 1.09–1.82). Furthermore, the affected family‐based control (AFBAC) association test and the transmission disequilibrium analysis of multiplex families of European descent from the British Diabetes Association (BDA) Warren Repository indicated that the association was stronger in −1123G > C compared to +1858C > T. In conclusion, the type 1 diabetes association with PTPN22 is confirmed, but it cannot be attributed solely to the +1858C > T variant. The promoter −1123G > C SNP is a more likely causative variant in PTPN22.

Enterovirus Infection, CXC Chemokine Ligand 10 (CXCL10), and CXCR3 Circuit: A Mechanism of Accelerated β-Cell Failure in Fulminant Type 1 Diabetes

OBJECTIVE Fulminant type 1 diabetes is characterized by the rapid onset of severe hyperglycemia and ketoacidosis, with subsequent poor prognosis of diabetes complications. Causative mechanisms for accelerated β-cell failure are unclear. RESEARCH DESIGN AND METHODS Subjects comprised three autopsied patients who died from diabetic ketoacidosis within 2–5 days after onset of fulminant type 1 diabetes. We examined islet cell status, including the presence of enterovirus and chemokine/cytokine/major histocompatibility complex (MHC) expressions in the pancreata using immunohistochemical analyses and RT-PCR. RESULTS Immunohistochemical analysis revealed the presence of enterovirus-capsid protein in all three affected pancreata. Extensive infiltration of CXCR3 receptor–bearing T-cells and macrophages into islets was observed. Dendritic cells were stained in and around the islets. Specifically, interferon-γ and CXC chemokine ligand 10 (CXCL10) were strongly coexpressed in all subtypes of islet cells, including β-cells and α-cells. No CXCL10 was expressed in exocrine pancreas. Serum levels of CXCL10 were increased. Expression of MHC class II and hyperexpression of MHC class I was observed in some islet cells. CONCLUSIONS These results strongly suggest the presence of a circuit for the destruction of β-cells in fulminant type 1 diabetes. Enterovirus infection of the pancreas initiates coexpression of interferon-γ and CXCL10 in β-cells. CXCL10 secreted from β-cells activates and attracts autoreactive T-cells and macrophages to the islets via CXCR3. These infiltrating autoreactive T-cells and macrophages release inflammatory cytokines including interferon-γ in the islets, not only damaging β-cells but also accelerating CXCL10 generation in residual β-cells and thus further activating cell-mediated autoimmunity until all β-cells have been destroyed.

Different contribution of class II HLA in fulminant and typical autoimmune type 1 diabetes mellitus

Aims/hypothesisFulminant type 1 diabetes, which is characterised by a markedly acute onset of diabetes and an absence of islet-related autoantibodies, accounts for 20% of type 1 diabetes in Japan. We aimed to clarify the contribution of the HLA subtype to fulminant type 1 diabetes in Japanese.MethodsWe determined the serological subtypes of HLA-A, -DR and -DQ in 115 patients with fulminant type 1 diabetes, 98 patients with typical type 1A diabetes and 190 normal control subjects.ResultsThe frequency of HLA-DR4, but not DR9, was significantly higher in fulminant type 1 diabetes, while those of HLA-DR1, DR2, DR5 and DR8 were significantly lower than those in controls. In contrast, DR9 but not DR4 was more frequent and DR2 was extremely rare in typical type 1A diabetes. Haplotype analysis revealed that DR4-DQ4 was significantly more frequent, and both DR2-DQ1 and DR8-DQ1 were less frequent in fulminant diabetes. In type 1A diabetes, DR2-DQ1 was extremely rare while DR9-DQ3 was significantly more frequent. In the combination analysis, the homozygotes of DR4-DQ4 in fulminant type 1 diabetes and DR9-DQ3 in typical type 1A diabetes indicated high odds ratios (13.3 and 13.3, respectively).Conclusions/interpretationOur results suggest that class II HLA contributes to the development of fulminant type 1 diabetes. Susceptibility and resistance of the HLA subtype to type 1 diabetes are distinct between fulminant and typical autoimmune type 1 diabetes.

Insulin Intervention in Slowly Progressive Insulin-Dependent (Type 1) Diabetes Mellitus

OBJECTIVE We tested the hypothesis that insulin therapy rather than sulfonylurea (SU) treatment is preferable to reverse or preserve beta-cell function among patients with slowly progressive insulin-dependent (type 1) diabetes (SPIDDM) or latent autoimmune diabetes in adults. METHODS This multicenter, randomized, nonblinded clinical study screened 4089 non-insulin-dependent diabetic patients for glutamic acid decarboxylase autoantibodies (GADAb). Sixty GADAb-positive non-insulin-requiring diabetic patients with a 5-yr duration or shorter of diabetes were assigned to either the SU group (n = 30) or the insulin group (n = 30). Serum C-peptide responses to annual oral glucose tolerance tests were followed up for a mean of 57 months. The primary endpoint was an insulin-dependent state defined by the sum of serum C-peptide values during the oral glucose tolerance test (SigmaC-peptide) less than 4 ng/ml (1.32 nmol/liter). RESULTS The progression rate to an insulin-dependent state in the insulin group (three of 30, 10%) was lower than that in the SU group (13 of 30, 43%; P = 0.003, log-rank). Longitudinal analysis demonstrated that SigmaC-peptide values were better preserved in the insulin group than in the SU group. Multiple regression analysis demonstrated that insulin treatment, a preserved C-peptide response, and a low GADAb titer at entry were independent factors in preventing progression to an insulin-dependent state. Subgroup analysis suggested that insulin intervention was highly effective for SPIDDM patients with high GADAb titers [> or =10 U/ml (180 World Health Organization U/ml)] and preserved beta-cell function [SigmaC-peptide > or = 10 ng/ml (3.31 nmol/liter)] at entry. No severe hypoglycemic episodes occurred during the study. CONCLUSIONS Insulin intervention to preserve beta-cell function is effective and safe for patients with SPIDDM or latent autoimmune diabetes in adults.

Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection

It has been reported that lactate dehydrogenase virus (LDV) selectively infects a subpopulation of macrophages, thereby affecting the immune system. We studied the effects of LDV infection on the development of diabetes in non-obese diabetic (NOD) mice. Five-week-old female NOD mice were infected with LDV (10(8) ID50/mouse) and observed until 23 weeks of age. None of the 21-LDV-infected mice developed diabetes, whereas 10/14 (71.4%) uninfected mice did. Although the subpopulations of T cells and the percentage of Mac1-positive cells in the NOD murine spleen and the number of harvested peritoneal macrophages were unaffected by LDV infection, the proportions of Ia-positive peritoneal macrophages were significantly decreased in LDV-infected compared with uninfected mice (1.1 +/- 0.2%, 6.5 +/- 2.9%; P < 0.01). In LDV-infected NOD mice, insulitis of the same grade as that seen in uninfected NOD mice was observed. In another experiment, 3, 5, 10 or 16-week-old female NOD mice were infected with LDV. None of the mice infected with LDV at 3, 5 or 10 weeks of age developed diabetes and only one of six infected at 16 weeks of age did. These findings indicate that LDV infection suppresses the development of diabetes in female NOD mice by reducing the capacity of Ia-positive macrophages, and suggest that the development of human type 1 diabetes may be suppressed by certain viral infections.

Vitamin D receptor initiation codon polymorphism influences genetic susceptibility to type 1 diabetes mellitus in the Japanese population

BackgroundVitamin D has been shown to exert manifold immunomodulatory effects. Type 1 diabetes mellitus (T1DM) is regarded to be immune-mediated and vitamin D prevents the development of diabetes in the NOD mouse. We studied the association between T1DM and the initiation codon polymorphism in exon 2 of the vitamin D receptor gene in a Japanese population. We also investigated associations between the vitamin D receptor polymorphism and GAD65-antibody (Ab) positivity. We carried out polymerase chain reaction-restriction fragment length polymorphism analysis in 110 Japanese T1DM patients and 250 control subjects. GAD65 antibodies were assessed in 78 patients with T1DM.ResultsWe found a significantly higher prevalence of the F allele / the FF genotype in the patients compared to the controls (P = 0.0069 and P = 0.014, respectively). Genotype and allele frequencies differed significantly between GAD65-Ab-positive patients and controls (P = 0.017 and P = 0.012, respectively), but neither between GAD65-Ab-negative patients and controls (P = 0.68 and P = 0.66, respectively) nor between GAD65-Ab-positive and -negative patients (P = 0.19 and P = 0.16, respectively).ConclusionsOur findings suggest that the vitamin D receptor initiation codon polymorphism influences genetic susceptibility to T1DM among the Japanese. This polymorphism is also associated with GAD65-Ab-positive T1DM, although the absence of a significant difference between GAD65-Ab-negative patients and controls might be simply due to the small sample size of patients tested for GAD65 antibodies.

Radioimmunoassay Detects the Frequent Occurrence of Autoantibodies to the Mr 65,000 Isoform of Glutamic Acid Decarboxylase in Japanese Insulin-Dependent Diabetes

Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.

Association of ceramides in human plasma with risk factors of atherosclerosis

Atherosclerosis is a multifactorial disorder. Recent studies indicate that the plasma level of sphingomyelin, which yields ceramide, correlates with the risk of coronary heart disease. Therefore, ceramide, a well-known lipid causing apoptosis in various cell types, may contribute to atherogenesis. We examined the relationship between ceramide concentration and risk factors of atherosclerosis in normal human plasma using electrospray tandem mass spectrometry (LC-MS/MS). Major ceramides in human plasma were C24∶0 and C24∶1. The ceramide concentration showed a significant positive correlation with total cholesterol (TC) and triglycerides (TG). In addition, plasma ceramide level increased drastically at a high level of LDL cholesterol (more than 170 mg/dL). Our previous studies demonstrated that the sum of fragmented and conjugated apolipoprotein B-100 proteins (B-ox), which were products of a radical reaction of LDL as well as plasma, was a reliable index of atherosclerosis. B-ox showed a significant positive correlation with the plasma ceramide level. Based on these results, we propose that the ceramide level in human plasma is a risk factor at the early stages of atherosclerosis.

Identification of mutations in the hepatocyte nuclear factor (HNF)-1 alpha gene in Japanese subjects with IDDM

One form of maturity-onset diabetes of the young, MODY3, is characterized by a severe insulin secretory defect, compared with MODY2, a glucokinase-deflcient diabetes. It has recently been shown that mutations of the gene encoding the transcription factor hepatocyte nuclear factor (HNF)-1α cause MODY3. Because of the rapid progress to overt diabetes and the high prevalence of required insulin treatment in patients with MODY3, we screened the HNF-1α gene for mutations in Japanese subjects with IDDM. Ten exons and flanking introns of the HNF-1α gene in these subjects were amplified by polymerase chain reaction and direct sequencing of the products. Mutations were identified in three (5.5%) of the 55 unrelated subjects with IDDM. A missense mutation of R272H (replacement of Arg by His in codon 272) in the DNA binding domain of HNF-1α was found in a subject who developed IDDM 1 year after diagnosis of NIDDM at 8 years of age. A frameshift mutation of P291fsinsC (insertion of a C in a polyC tract around codon 291 for Pro), which would generate a mutant truncated protein of 340 amino acids, was found in a subject who started insulin treatment when hyperglycemia and ketonuria were noticed at 13 years of age. A missense mutation of R583G (replacement of Arg by Gly in codon 583) in the transactivation domain of HNF-1α was found in a subject with sudden-onset IDDM at 20 years of age. None of these mutations were present in 100 nondiabetic subjects (200 normal chromosomes). These results indicate that the HNF-1α gene defects could lead to the development of not only early-onset NIDDM but also IDDM, implicating the importance of subclassification of HNF-1α-deficient IDDM from a classical type of autoimmune-based IDDM in Japanese.

Insulin Intervention to Preserve β Cells in Slowly Progressive Insulin‐Dependent (Type 1) Diabetes Mellitusa

Abstract: Slowly progressive insulin‐dependent (type 1) diabetes mellitus (SPIDDM) is characterized by (1) late age of onset, with initial features of NIDDM and subsequent progression to insulin‐dependent stage; (2) high predictive value of autoantibodies against glutamic acid decarboxylase (GADAb) and islet cell antibodies (ICA) for progression of β cell failure; (3) less predominant T cell response, which may attack and eventually destroy β‐cells in affected pancreas. These findings may suggest a rationale for intervention to prevent slowly progressive β cell dysfunction in this type of diabetes. We identified three independent risk factors for progression of β cell failure in SPIDDM: (1) sulfonylurea treatment; (2) ICA‐positive periods; and (3) initial body weight. We hypothesized that removal of the risk factors for further progression of β cell dysfunction will have beneficial effects on intervention strategy in treating SPIDDM. In our pilot study, we used a small dose of insulin instead of sulfonylurea in the early stage of treatment of patients with SPIDDM. Insulin‐treated SPIDDM patients had a sustained C peptide response (CPR), while most of sulfonylurea‐treated patients progressed to an insulin‐dependent state. We organized a randomized multicenter clinical trial to study early treatment to prevent the progression of β cell dysfunction in SPIDDM (the Tokyo Study). It was demonstrated that early intervention with insulin therapy is an effective treatment modality in the early stage of SPIDDM patients who had preserved β cell function at entry (integrated value of serum C peptide values at 0, 30, 60, 90, and 120 minutes; Sigma CPR ≥ 10 ng/mL) and high GADAb (>10 U/mL). Preventive insulin treatment was ineffective in the patients who had diminished insulin reserve at entry (Sigma CPR < 10 ng/mL). Insulin intervention to preserve β cell dysfunction in SPIDDM is effective and safe in patients with preserved β cell function and high GADAb titers at the initiation of insulin.