Shoichiro Tanaka

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.

Corticosteroid-responsive diabetes mellitus associated with autoimmune pancreatitis

Autoimmune pancreatitis, which can be treated with corticosteroid therapy, has the potential to induce diabetes. In a cohort study of 2220 patients with suspected chronic pancreatitis, we found that all four patients with autoimmune chronic pancreatitis also had diabetes. Treatment with prednisolone subsequently improved insulin secretion and glycaemic control in these patients.

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.

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.

Amylase α-2A Autoantibodies: Novel Marker of Autoimmune Pancreatitis and Fulminant Type 1 Diabetes

OBJECTIVE— The pathogenesis of autoimmune pancreatitis (AIP) and fulminant type 1 diabetes remains unclear, although it is known that immune-mediated processes severely compromise the endocrine and exocrine functions in both diseases. RESEARCH DESIGN AND METHODS— We have screened a λTriplEx2 human pancreas cDNA library with serum from a patient with AIP and obtained positive clones. Sequence analysis revealed that 7 of 10 clones were identical to human amylase α-2A. Using a recombinant COOH-terminal amylase α-2A protein, we developed an enzyme-linked immunosorbent assay system to detect autoantibodies against human amylase α-2A. RESULTS— All 15 serum samples from patients with AIP recognized the recombinant protein, whereas sera from 25 patients with chronic alcoholic pancreatitis and sera from 25 patients with a pancreas tumor did not. Interestingly, 88% (15/17) of patients with fulminant type 1 diabetes were positive for an autoantibody against amylase α-2A. These antibodies were detected in 21% of patients with acute-onset type 1 diabetes (9 of 42) and 6% of type 2 diabetic patients (4 of 67). CONCLUSIONS— These results suggest that an autoantibody against amylase α-2A is a novel diagnostic marker for both AIP and fulminant type 1 diabetes and that, clinically and immunologically, AIP and fulminant type 1 diabetes are closely related.OBJECTIVE The pathogenesis of autoimmune pancreatitis (AIP) and fulminant type 1 diabetes remains unclear, although it is known that immune-mediated processes severely compromise the endocrine and exocrine functions in both diseases. RESEARCH DESIGN AND METHODS We have screened a lambdaTriplEx2 human pancreas cDNA library with serum from a patient with AIP and obtained positive clones. Sequence analysis revealed that 7 of 10 clones were identical to human amylase alpha-2A. Using a recombinant COOH-terminal amylase alpha-2A protein, we developed an enzyme-linked immunosorbent assay system to detect autoantibodies against human amylase alpha-2A. RESULTS All 15 serum samples from patients with AIP recognized the recombinant protein, whereas sera from 25 patients with chronic alcoholic pancreatitis and sera from 25 patients with a pancreas tumor did not. Interestingly, 88% (15/17) of patients with fulminant type 1 diabetes were positive for an autoantibody against amylase alpha-2A. These antibodies were detected in 21% of patients with acute-onset type 1 diabetes (9 of 42) and 6% of type 2 diabetic patients (4 of 67). CONCLUSIONS These results suggest that an autoantibody against amylase alpha-2A is a novel diagnostic marker for both AIP and fulminant type 1 diabetes and that, clinically and immunologically, AIP and fulminant type 1 diabetes are closely related.

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.

RIG-I– and MDA5-Initiated Innate Immunity Linked With Adaptive Immunity Accelerates β-Cell Death in Fulminant Type 1 Diabetes

OBJECTIVE The contribution of innate immunity responsible for aggressive β-cell destruction in human fulminant type 1 diabetes is unclear. RESEARCH DESIGN AND METHODS Islet cell expression of Toll-like receptors (TLRs), cytoplasmic retinoic acid–inducible gene I (RIG-I)-like receptors, downstream innate immune markers, adaptive immune mediators, and apoptotic markers was studied in three autopsied pancreata obtained 2 to 5 days after onset of fulminant type 1 diabetes. RESULTS RIG-I was strongly expressed in β-cells in all three pancreata infected with enterovirus. Melanoma differentiation–associated gene-5 was hyperexpressed in islet cells, including β- and α-cells. TLR3 and TLR4 were expressed in mononuclear cells that infiltrated islets. Interferon (IFN)-α and IFN-β were strongly expressed in islet cells. Major histocompatibility complex (MHC)-class I, IFN-γ, interleukin-18, and CXC motif ligand 10 were expressed and colocalized in affected islets. CD11c+ MHC-class II+ dendritic cells and macrophage subsets infiltrated most islets and showed remarkable features of phagocytosis of islet cell debris. CD4+ forkhead box P3+ regulatory T cells were not observed in and around the affected islets. Mononuclear cells expressed the Fas ligand and infiltrated most Fas-expressing islets. Retinoic acid–receptor responder 3 and activated caspases 8, 9, and 3 were preferentially expressed in β-cells. Serum levels of IFN-γ were markedly increased in patients with fulminant type 1 diabetes. CONCLUSIONS These findings demonstrate the presence of specific innate immune responses to enterovirus infection connected with enhanced adoptive immune pathways responsible for aggressive β-cell toxicity in fulminant type 1 diabetes.

HSP 10 is a new autoantigen in both autoimmune pancreatitis and fulminant type 1 diabetes.

To search autoantigens in autoimmune pancreatitis (AIP), we have screened the human pancreas cDNA library with a patients serum and obtained 10 positive clones. Seven out of 10 clones were amylase alpha-2A, the autoantibody to which was specifically detected in sera from patients with AIP and fulminant type 1 diabetes (FT1DM) [T. Endo, S. Takizawa, S. Tanaka, M. Takahashi, H. Fujii, T. Kamisawa, T. Kobayashi, Amylase alpha-2A autoantibodies: novel marker of autoimmune pancreatitis and fulminant type 1 diabetes mellitus, Diabetes 58 (2009) 732-737]. Sequencing of 1 out of remaining 3 positive clones revealed that it was identical to heat shock protein 10 (HSP 10) cDNA. Using a recombinant HSP 10, we have developed enzyme-linked immunosorbent assay (ELISA) system for detecting autoantibodies against HSP 10. We found that autoantibody against HSP 10 was also produced with high frequency in sera from patients with AIP (92%) and FT1DM (81%), but not in chronic alcoholic pancreatitis (8%) or healthy volunteers (1.4%). These results suggest that an autoantibody against HSP 10 is also a new diagnostic marker for both AIP and FT1DM.

Corticosteroid-responsive diabetes mellitus associated with autoimmune pancreatitis: pathological examinations of the endocrine and exocrine pancreas.

Abstract: Histopathological changes of biopsied pancreata were examined in three diabetic patients with autoimmune chronic pancreatitis (ACP). We found intra‐ and periinsular mononuclear cell (MNC)—mainly CD8+ T cell—infiltration as well as MNC infiltration around the ductal cells. Islet β cell volume decreased significantly. Some ductal cells expressed insulin and insulin promoter factor‐1 concomitantly in the affected pancreas. These findings provide new insight toward understanding the pathological mechanisms of corticosteroid treatment‐responsive diabetes associated with ACP.

Genetic Association between the Interleukin-2 Receptor-α Gene and Mode of Onset of Type 1 Diabetes in the Japanese Population

CONTEXT/OBJECTIVE The IL-2 receptor-alpha (IL2RA), also known as CD25, is expressed on the regulatory T cells, which play an important role in the control of immune responses and the maintenance of immune homeostasis. Our objective was to determine whether variants in the IL2RA gene are associated with type 1 diabetes in the Japanese population. DESIGN/PATIENTS We genotyped the four single-nucleotide polymorphisms (rs706778, rs3118470, ss52580101, and rs11594656) of the IL2RA in 885 patients with type 1 diabetes and 606 control subjects of Japanese origin. The allele and genotype frequencies were examined in the patient groups stratified by their mode of onset in a case-control study. RESULTS We found evidence of association with acute-onset, but not slow-onset and fulminant, type 1 diabetes for two of the four single-nucleotide polymorphisms genotyped (rs706778 and rs3118470). The rs706778 A allele and the rs3118470 G allele were associated with an increased disease risk [odds ratio (OR) for rs706778 AA genotype 1.54, P = 4.2 x 10(-4) and OR for rs3118470 GG genotype 1.50, P = 0.0019, respectively]. Furthermore, the A-G haplotype was associated with increased type 1 diabetes risk in the acute-onset form (OR 1.30, P = 0.002). CONCLUSIONS The present data confirm the type 1 diabetes association with IL2RA and provide evidence that the different contributions of the IL2RA in the susceptibility to acute-onset and other forms of type 1 diabetes in the Japanese population.