Tyrosine Phosphatase-like Protein Research Articles

Production and antigenicity analysis of a recombinant insulinoma associated protein-2 in HEK293 cells

Insulinoma-associated protein-2 (IA-2) is a transmembrane glycoprotein belonging to the tyrosine phosphatase-like protein family as well as an important autoantigen in the diagnosis of type 1 diabetes. IA-2 products have been marketed in Europe and the United States. At present, commercially available IA-2 antigens are either the recombinant IA-2ic domain or the IA-2 naturally extracted from bovine islets. However, the recombinant IA-2 antigen displays weak positive in clinic practice, which often results in occasional detection failures, thus cannot completely replace the naturally extracted IA-2 antigen. In this study, an HEK293 expression system was used to explore the production of recombinant IA-2. An IA-2 transmembrane fragment (IA-2 TMF) located at amino acid position 449-979, also known as the natural membrane protein form of IA-2, was produced in HEK293 through transfection, and both the expression conditions and dissolution conditions of the membrane protein were also optimized. The purified membrane protein yield was 0.78 mg/L cell culture. Subsequently, the antigen activity of IA-2 TMF was compared with RSR rhIA-2 through enzyme linked immunosorbent assay. The serum of 77 type 1 diabetes patients and 32 healthy volunteers were detected. Receiver operating characteristic curve (ROC) curve was used to characterize the sensitivity and specificity of the test results. The results showed that the sensitivity of IA-2 TMF was 71.4% (55/77), while the sensitivity of RSR rhIA-2 was 63.6% (49/77), and the specificity of both antigens were all 100%. There was no significant difference in specificity between the two antigens, but the sensitivity of IA-2 TMF was appreciably better than that of the imported gold standard RSR rhIA-2 antigen. In conclusion, the recombinant IA-2 TMF produced in HEK293 cells can be used as a raw material to develop in vitro diagnostic reagents for type 1 diabetes.

Anti-Islet Autoantibodies in Type 1 Diabetes.

Anti-islet autoantibodies serve as key markers in immune-mediated type 1 diabetes (T1D) and slowly progressive T1D (SPIDDM), also known as latent autoimmune diabetes in adults (LADA). Autoantibodies to insulin (IAA), glutamic acid decarboxylase (GADA), tyrosine phosphatase-like protein IA-2 (IA-2A), and zinc transporter 8 (ZnT8A) are currently employed in the diagnosis, pathological analysis, and prediction of T1D. GADA can also be detected in non-diabetic patients with autoimmune diseases other than T1D and may not necessarily reflect insulitis. Conversely, IA-2A and ZnT8A serve as surrogate markers of pancreatic β-cell destruction. A combinatorial analysis of these four anti-islet autoantibodies demonstrated that 93-96% of acute-onset T1D and SPIDDM cases were diagnosed as immune-mediated T1D, while the majority of fulminant T1D cases were autoantibody-negative. Evaluating the epitopes and immunoglobulin subclasses of anti-islet autoantibodies help distinguish between diabetes-associated and non-diabetes-associated autoantibodies and is valuable for predicting future insulin deficiency in SPIDDM (LADA) patients. Additionally, GADA in T1D patients with autoimmune thyroid disease reveals the polyclonal expansion of autoantibody epitopes and immunoglobulin subclasses. Recent advancements in anti-islet autoantibody assays include nonradioactive fluid-phase assays and the simultaneous determination of multiple biochemically defined autoantibodies. Developing a high-throughput assay for detecting epitope-specific or immunoglobulin isotype-specific autoantibodies will facilitate a more accurate diagnosis and prediction of autoimmune disorders. The aim of this review is to summarize what is known about the clinical significance of anti-islet autoantibodies in the pathogenesis and diagnosis of T1D.

Insights on the role of anti-inflammatory and immunosuppressive agents in the amelioration of diabetes.

Diabetes is a major health problem worldwide. It is a chronic metabolic disorder that produces overt hyperglycemic condition that occurs either when the pancreas does not produce enough insulin due to excessive destruction of pancreatic β-cells (type 1 diabetes) or due to development of insulin resistance (type 2 diabetes). An autoimmune condition known as type 1 diabetes (T1D) results in the targeted immune death of β-cells that produce insulin. The only available treatment for T1D at the moment is the lifelong use of insulin. Multiple islet autoantibody positivity is used to diagnose T1D. There are four standard autoantibodies observed whose presence shows the development of T1D: antibodies against insulin, glutamic acid decarboxylase (GAD65), zinc T8 transporter (ZnT8), and tyrosine phosphatase-like protein (ICA512). In type 2 diabetes (T2D), an inflammatory response precipitates as a consequence of the immune response to high blood glucose level along with the presence of inflammation mediators produced by macrophages and adipocytes in fat tissue. The slow and chronic inflammatory condition of adipose tissue produces insulin resistance leading to increased stress on pancreatic β-cells to produce more insulin to compensate for the insulin resistance. Thus, this stress condition exacerbates the apoptosis of β-cells leading to insufficient production of insulin, resulting in hyperglycemia which signifies late stage T2D. Therefore, the therapeutic utilization of immunosuppressive agents may be a better alternative over the use of insulin and oral hypoglycemic agents for the treatment of T1D and T2D, respectively. This review enlightens the immune intervention for the prevention and amelioration of T1D and T2D in humans with main focus on the antigen-specific immune suppressive therapy.

Physical activity and progression to type 1 diabetes in children and youth with islet autoimmunity: The diabetes autoimmunity study in the young.

Physical inactivity may contribute to islet autoimmunity and progression to clinical type 1 diabetes. To test this hypothesis, we evaluated physical activity, assessed by accelerometer, as an independent risk factor for progression to clinical diabetes among genetically at risk for type 1 diabetes children and youth with islet autoimmunity. Accelerometer data were obtained for 95 children and youth participating in the diabetes autoimmunity study in the young who had islet autoimmunity. Islet autoimmunity was defined as the presence of islet autoantibodies to insulin, glutamic acid decarboxylase, tyrosine phosphatase-like protein IA-2, or zinc transporter 8. During prospective follow-up for up to 7 years, 13 of the 95 participants progressed to clinical diabetes. In multivariable survival analysis, none of the physical activity parameters examined predicted a higher risk of developing diabetes. In survival analysis with time-varying physical activity parameters, none of the physical activity parameters over time were associated with the risk of developing type 1 diabetes. It does not appear that low-physical activity is a risk factor for progression from islet autoantibodies to diabetes in children and youth at high-genetic risk for type 1 diabetes.

Paladin, a tyrosine phosphatase-like protein, is required for XA21-mediated immunity in rice

XA21 encodes a rice immune receptor that confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo). XA21-mediated immunity is triggered by recognition of a small protein called RaxX-sY (required for activation of XA21-mediated immunity X, tyrosine-sulfated) secreted by Xoo. To identify components regulating XA21-mediated immunity, we generated and screened a mutant population of fast-neutron-mutagenized rice expressing Ubi:Myc-XA21 for those susceptible to Xoo. Here, we report the characterization of one of these rice mutants, named sxi2 (suppressor of XA21-mediated immunity-2). Whole-genome sequencing revealed that sxi2 carries a deletion of the PALADIN (PALD) gene encoding a protein with three putative protein tyrosine phosphatase-like domains (PTP-A, -B, and -C). Expression of PALD in the sxi2 genetic background was sufficient to complement the susceptible phenotype, which requires the catalytic cysteine of the PTP-A active site to restore resistance. PALD co-immunoprecipitated with the full-length XA21 protein, whose levels are positively regulated by the presence of the PALD transgene. Furthermore, we foundd that sxi2 retains many hallmarks of XA21-mediated immunity, similar to the wild type. These results reveal that PALD, a previously uncharacterized class of phosphatase, functions in rice innate immunity, and suggest that the conserved cysteine in the PTP-A domain of PALD is required for its immune function.

ICA512 RESP18 homology domain is a protein-condensing factor and insulin fibrillation inhibitor

Type 1 diabetes islet cell autoantigen 512 (ICA512/IA-2) is a tyrosine phosphatase-like intrinsic membrane protein involved in the biogenesis and turnover of insulin secretory granules (SGs) in pancreatic islet β-cells. Whereas its membrane-proximal and cytoplasmic domains have been functionally and structurally characterized, the role of the ICA512 N-terminal segment named "regulated endocrine-specific protein 18 homology domain" (RESP18HD), which encompasses residues 35-131, remains largely unknown. Here, we show that ICA512 RESP18HD residues 91-131 encode for an intrinsically disordered region (IDR), which in vitro acts as a condensing factor for the reversible aggregation of insulin and other β-cell proteins in a pH and Zn2+-regulated fashion. At variance with what has been shown for other granule cargoes with aggregating properties, the condensing activity of ICA512 RESP18HD is displayed at a pH close to neutral, i.e. in the pH range found in the early secretory pathway, whereas it is resolved at acidic pH and Zn2+ concentrations resembling those present in mature SGs. Moreover, we show that ICA512 RESP18HD residues 35-90, preceding the IDR, inhibit insulin fibrillation in vitro Finally, we found that glucose-stimulated secretion of RESP18HD upon exocytosis of SGs from insulinoma INS-1 cells is associated with cleavage of its IDR, conceivably to prevent its aggregation upon exposure to neutral pH in the extracellular milieu. Taken together, these findings point to ICA512 RESP18HD being a condensing factor for protein sorting and granulogenesis early in the secretory pathway and for prevention of amyloidogenesis.

SUN-170 Development of Type 1 Diabetes Mellitus on Immune Checkpoint Inhibitor

Introduction: Immune checkpoint inhibitors (ICPi) are used to augment the immune response and have led to improved outcomes in various malignancies. With this enhanced immunologic response comes a spectrum of side effects, known as immune-related adverse events (iRAEs). These are most commonly dermatologic, gastrointestinal, and endocrine-related events, with development of autoimmune diabetes occurring in only 0.2% of all reported iRAEs. Case: A 61-year-old male with pre-diabetes and metastatic small cell lung cancer post chemotherapy previously on combination ICPi, nivolumab and ipilimumab, presented with worsening nausea, vomiting, and decreased oral intake. He received 4 doses of combined therapy, developed presumed secondary adrenal insufficiency, and was switched to ipilimumab monotherapy for 10 doses. He started to have hyperglycemia within the first month of being on monotherapy. Despite discontinuation of ipilimumab 2 months prior to presentation, he had progressive hyperglycemia, which was poorly controlled with glipizide. At initial presentation, laboratory findings revealed diabetic ketoacidosis (DKA). He was managed on an insulin drip with resolution within 24 hrs. Workup showed a hemoglobin A1c of 7.9%, undetectable c-peptide level and negative autoimmune diabetes antibodies. He was diagnosed with new onset type 1 diabetes (T1DM) and was discharged on basal-bolus insulin. Discussion: The most common adverse events with ICPi use are diarrhea, fatigue, pruritus, and rash. While the most common endocrinopathies reported are thyroid dysfunction and hypophysitis, providers should also be aware of the serious but rarer iRAEs of autoimmune adrenalitis and autoimmune diabetes. All but one reported case is associated with the use of PD-1 inhibitor monotherapy or combination therapy with CTLA-4 inhibitor. According to a recent meta-analysis, around 85.7% of cases presented in DKA within 1 to 52 weeks following ICPi initiation and, like our patient, usually have mildly elevated hemoglobin A1c’s and low levels of c-peptide. Individuals with T1DM can express any combination of islet cell antibodies (ICA), insulin autoantibodies (IAA), antibodies to glutamic acid decarboxylase (GAD), antibodies to tyrosine phosphatase-like proteins (IA-2, ICA512), and antibodies to zinc transporter 8 (ZnT8). No dominant pattern of antibody production is observed in the reported cases of ICPi-associated T1DM. Although these antibodies can help in diagnosing T1DM, more than 25% lack such autoantibodies and are still classified as having T1DM, as seen in this case. Conclusion: ICPi can potentially prolong survival for many cancer patients with an otherwise grim prognosis. As ICPi use is expected to expand in the coming years, it is especially important to study and report less common iRAEs to better understand, recognize, and manage these potentially serious adverse effects.

Phosphatidylglycerophosphate phosphatase is required for root growth in Arabidopsis

Phosphatidylglycerol (PG) is an indispensable lipid class in photosynthetic activity. However, the importance of PG biosynthesis in non-photosynthetic organs remains elusive. We previously identified phosphatidylglycerophosphate phosphatase 1 (PGPP1), which catalyzes the last step of PG biosynthesis in Arabidopsis thaliana. In the present report, we noted considerably shorter roots of the pgpp1-1 mutant compared to the wild type. We observed defective order of columella cells in the root apices, which was complemented by introducing the wild-type PGPP1 gene. Although PGPP1 is chloroplast-localized in leaf mesophyll cells, we observed mitochondrial localization of PGPP1 in root cells, suggesting possible dual targeting of PGPP1. Moreover, we identified previously uncharacterized 2 protein tyrosine phosphatase-like proteins as functional PGPPs. These proteins, designated PTPMT1 and PTPMT2, complemented growth and lipid phenotypes of Δgep4, a Saccharomyces cerevisiae mutant of PGPP. The ptpmt1-1 ptpmt2-1 exhibited no visible phenotype; however, the pgpp1-1 ptpmt1-1 ptpmt2-1 significantly enhanced the root phenotype of pgpp1-1 without further affecting the photosynthesis, suggesting that these newly found PGPPs are involved in the root phenotype. Radiolabeling experiment of mutant roots showed that decreased PG biosynthesis is associated with the mutation of PGPP1. These results suggest that PG biosynthesis is required for the root growth.

Multiple organ-specific autoantibodies in patients with phenotypic type 2 diabetes in China: a post-hoc analysis of the China National Diabetes and Metabolic Disorders Study

BackgroundIn the China National Diabetes and Metabolic Disorders Study, age-standardised prevalence of total diabetes was 9·7%, accounting for 92·4 million adults, in China in 2010. Using clinical data and serum samples from this study, we aimed to investigate the prevalence of several organ-specific autoantibodies in patients with phenotypic type 2 diabetes. MethodsThis study included a nationally representative sample of 46 239 adults aged 20 years or older from 14 provinces and municipalities. After an overnight fast, participants had an oral glucose tolerance test. Previously diagnosed diabetes was self-reported. We randomly selected 4671 patients with diabetes and 1002 individuals with normal glucose tolerance as controls. Participants were screened centrally for glutamic acid decarboxylase autoantibodies (GAD-Ab), protein tyrosine phosphatase-like protein autoantibodies (IA2-Ab), and zinc transporter isoform 8 autoantibodies (ZnT8-Ab) using validated methods of the Islets Autoantibody Standardization Program 2012. We then assayed for thyroid peroxidase autoantibodies (TPO-Ab), tissue transglutaminase autoantibodies (tTG-Ab), and 21-hydroxylase autoantibodies (21-OH-Ab) in patients with islet autoantibody-positive diabetes (n=175), in randomly selected age-matched and sex-matched patients with islet autoantibody-negative diabetes (n=3052 for TPO-Ab, n=525 for tTG-Ab, and n=525 for 21-OH-Ab), and in individuals with normal glucose tolerance (n=954 for TPO-Ab, n=200 for tTG-Ab, and n=200 for 21-OH-Ab). We used multiple logistic regression analysis to determine the risk factor of each autoantibody. Ethics approval was granted by the Second Xiangya Hospital of Central South University, and all participants provided written informed consent. FindingsAfter adjustment for age and sex using results from the national population census, the standardised prevalence of autoimmune diabetes was 6·0% in all patients with diabetes, which corresponded to about 6 million adults in China. In patients with diabetes, the adjusted antibody positivity was 3·6% for GAD-Ab, 1·2% for IA2-Ab, and 1·5% for ZnT8-Ab. In those with autoimmune diabetes, the adjusted antibody positivity was 16·3% for TPO-Ab, 2·1% for tTG-Ab, and 1·8% for 21-OH-Ab. In patients with islet autoantibody-negative diabetes, the adjusted positivity was 10·2% for TPO-Ab, 0·3% for tTG-Ab, and 0·5% for 21-OH-Ab. In Chinese adults with autoimmune diabetes, those positive for GAD-Ab had a high risk for TPO-Ab positivity (odds ratio 2·39, 95% CI 1·35–4·24; p=0·0031) and tTG-Ab positivity (6·983, 1·24–39·22; p=0·027), and those positive for IA2-Ab had a high risk for tTG-Ab positivity (19·05, 3·14–115·61; p=0·0010). Patients positive for TPO-Ab were at high risk of being positive for 21-OHAb (7·48, 1·44–38·74; p=0·016) and vice versa (7·21, 1·35–38·45; p=0·021). Of note, women had a high risk for TPO-Ab positivity compared with men (3·83, 2·20–6·68; p<0·0001). InterpretationPatients positive for islet autoantibodies have a high risk for other organ-specific autoimmune diseases. Screening of TPO-Ab and tTG-Ab in Chinese individuals with autoimmune diabetes should be considered a priority. FundingNational Key Technology R&D Program (2012BAI02B04), the Grant of National Clinical Research Center for Metabolic Diseases (2013BAI09B12), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1195), and the Key Project of Chinese Ministry of Education (113050A).

Sugar intake is associated with progression from islet autoimmunity to type 1 diabetes: the Diabetes Autoimmunity Study in the Young.

Dietary sugar intake may increase insulin production, stress the beta cells and increase the risk for islet autoimmunity (IA) and subsequent type 1 diabetes. Since 1993, the Diabetes Autoimmunity Study in the Young (DAISY) has followed children at increased genetic risk for type 1 diabetes for the development of IA (autoantibodies to insulin, GAD or protein tyrosine phosphatase-like protein [IA2] twice or more in succession) and progression to type 1 diabetes. Information on intake of fructose, sucrose, total sugars, sugar-sweetened beverages, beverages with non-nutritive sweetener and juice was collected prospectively throughout childhood via food frequency questionnaires (FFQs). We examined diet records for 1,893 children (mean age at last follow-up 10.2 years); 142 developed IA and 42 progressed to type 1 diabetes. HLA genotype was dichotomised as high risk (HLA-DR3/4,DQB1*0302) or not. All Cox regression models were adjusted for total energy, FFQ type, type 1 diabetes family history, HLA genotype and ethnicity. In children with IA, progression to type 1 diabetes was significantly associated with intake of total sugars (HR 1.75, 95% CI 1.07-2.85). Progression to type 1 diabetes was also associated with increased intake of sugar-sweetened beverages in those with the high-risk HLA genotype (HR 1.84, 95% CI 1.25-2.71), but not in children without it (interaction p value = 0.02). No sugar variables were associated with IA risk. Sugar intake may exacerbate the later stage of type 1 diabetes development; sugar-sweetened beverages may be especially detrimental to children with the highest genetic risk of developing type 1 diabetes.

Trimming of two major type 1 diabetes driving antigens, GAD65 and IA-2, allows for successful expression in Lactococcus lactis.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterised by excessive immune reactions against auto-antigens of pancreatic β-cells. Restoring auto-antigen tolerance remains the superior therapeutic strategy. Oral auto-antigen administration uses the tolerogenic nature of the gut-associated immune system to induce antigen-specific tolerance. However, due to gastric degradation, proper mucosal product delivery often imposes a challenge. Recombinant Lactococcus lactis have proven to be effective and safe carriers for gastrointestinal delivery of therapeutic products: L. lactis secreting diabetes-associated auto-antigens in combination with interleukin (IL)-10 have demonstrated therapeutic efficacy in a well-defined mouse model for T1D. Here, we describe the construction of recombinant L. lactis secreting the 65 kDa isoform of glutamic acid decarboxylase (GAD65) and tyrosine phosphatase-like protein ICA512 (IA-2), two major T1D-related auto-antigens. Attempts to secrete full size human GAD65 and IA-2 protein by L. lactis were unsuccessful. Trimming of GAD65 and IA-2 was investigated to optimise antigen secretion while maintaining sufficient bacterial growth. GAD65370-575 and IA-2635-979 showed to be efficiently secreted by recombinant L. lactis. Antigen secretion was verified by immunoblotting. Plasmid-derived GAD65 and IA-2 expression was combined in single strains with human IL-10 expression, a desired combination to allow tolerance induction. This study reports the generation of recombinant L. lactis secreting two major diabetes-related auto-antigens: human GAD65 and IA-2, by themselves or combined with the anti-inflammatory cytokine human IL-10. Prohibitive sequence obstacles hampering antigen secretion were resolved by trimming the full size proteins.

Correction: A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

Correction: A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

Type 1 diabetes (T1D) results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As) are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA) based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

Administering 25-hydroxyvitamin D3 in vitamin D-deficient young type 1A diabetic patients reduces reactivity against islet autoantigens

We investigated whether improving 25-hydroxyvitamin D status in young type 1A diabetic patients reduces reactivity of peripheral blood mononuclear cells against islet autoantigens and associates with beta-cell functional changes. Eight patients with 25-hydroxyvitamin D deficiency (<20ng/ml), out of 15 consecutive young type 1A diabetic subjects received 25-hydroxyvitamin D3 to achieve and maintain levels above 50ng/ml for up to one year. Peripheral blood mononuclear cell reactivity (Interferon-γ spots) against beta-cell autoantigens (glutamic acid decarboxylase 65-kD isoform, proinsulin and tyrosine phosphatase-like protein IA-2) and C-peptide during mixed meal were assessed before and after 25-hydroxyvitamin D3 replenishment. Target 25-hydroxyvitamin D blood levels were safely reached and maintained. Peripheral blood mononuclear cell reactivity against glutamic acid decarboxylase 65-kD isoform (3.8±4.0 vs. 45±16) and proinsulin (3.5±3.2 vs. 75±51) decreased significantly (p<0.001 and p<0.02) upon 25-hydroxyvitamin D3 replenishment, which was correlated with 25-hydroxyvitamin D concentrations. C-peptide values remained stable after one year of treatment. Safely restored and maintained 25-hydroxyvitamin D levels associated with reduced peripheral blood mononuclear cell reactivity against beta-cell autoantigens with no significant decrease of beta-cell function in this cohort of patients.

Clinical characteristics and autoantibody pattern in newly diagnosed adult‑onset autoimmune diabetes

Autoimmune diabetes in adults comprises a broad spectrum of clinical phenotypes. The aim of the study was to investigate clinical and biochemical features and anti-islet autoantibody pattern in adult patients with newly diagnosed autoimmune diabetes with regard to age and the number of autoantibodies detected at diagnosis. We retrospectively evaluated 344 patients (aged ≥18 years) with newly diagnosed diabetes and a positive anti-islet antibody titer. Patients were divided based on age (<35 and ≥35 years of age) or the number of detected autoantibodies (1, 2, or 3). The studied age groups did not differ with respect to the majority of clinical and laboratory features (e.g., clinical presentation, metabolic status, or degree of insulin deficiency). Autoantibodies to islet cell cytoplasm and to glutamic acid decarboxylase 65 occurred more frequently in younger patients, while the prevalence of autoantibodies to intracytoplasmatic domain of the tyrosine phosphatase-like protein (IA-2A) was similar in both age groups. Single autoantibody positivity was observed more often in older patients. The most common isolated autoantibody in this group was IA-2A. The presence of multiple autoantibodies was associated with younger age, lower fasting and stimulated C-peptide levels, and shorter duration of symptoms. The patient's age at diabetes onset does not determine clinical and biochemical characteristics at diagnosis but is associated with different autoantibody status. IA-2A antibodies may be useful in diagnosing autoimmune diabetes in adult patients. The assessment of the immune profile at diagnosis may help identify patients at a higher risk of significant insulin deficiency.

Prevalence of monogenic diabetes in the population-based Norwegian Childhood Diabetes Registry

Monogenic diabetes (MD) might be misdiagnosed as type 1 diabetes. The prevalence of MD among children with apparent type 1 diabetes has not been established. Our aim was to estimate the prevalence of common forms of MD in childhood diabetes. We investigated 2,756 children aged 0-14years with newly diagnosed diabetes who had been recruited to the nationwide population-based Norwegian Childhood Diabetes Registry (NCDR), from July 2002 to March 2012. Completeness of ascertainment was 91%. Children diagnosed with diabetes who were under12months of age were screened for mutations in KCNJ11, ABCC8 and INS. Children without GAD and protein tyrosine phosphatase-like protein antibodies were screened in two ways. Those who had a parent with diabetes were screened for mutations in HNF1A, HNF4A, INS and MT-TL1. Children with HbA1c <7.5% (<58mmol/mol) and no insulin requirement were screened for mutations in GCK. Finally, we searched the Norwegian MODY Registry for children with genetically verified MD. We identified 15 children harbouring a mutation in HNF1A, nine with one in GCK, four with one in KCNJ11, one child with a mutation in INS and none with a mutation in MT-TL1. The minimum prevalence of MD in the NCDR was therefore 1.1%. By searching the Norwegian MODY Registry, we found 24 children with glucokinase-MODY, 15 of whom were not present in the NCDR. We estimated the minimum prevalence of MD among Norwegian children to be 3.1/100,000. This is the first prevalence study of the common forms of MD in a nationwide, population-based registry of childhood diabetes. We found that 1.1% of patients in the Norwegian Childhood Diabetes Registry had MD.

Obesity-Associated Autoantibody Production Requires AIM to Retain the Immunoglobulin M Immune Complex on Follicular Dendritic Cells

Natural immunoglobulin M (IgM) is reactive to autoantigens and is believed to be important for autoimmunity. Blood pentameric IgM loaded with antigens forms a large immune complex (IC) that contains various elements, including apoptosis inhibitor of macrophage (AIM). Here we demonstrate that this IgM-AIM association contributes to autoantibody production under obese conditions. In mice fed a high-fat diet, natural IgM increased through B cell TLR4 stimulation. AIM associated with IgM and protected AIM from renal excretion, increasing blood AIM levels along with the obesity-induced IgM augmentation. Meanwhile, the AIM association inhibited IgM binding to the Fcα/μ receptor on splenic follicular dendritic cells, thereby protecting the IgM IC from Fcα/μ receptor-mediated internalization. This supported IgM-dependent autoantigen presentation to B cells, stimulating IgG autoantibody production. Accordingly, in obese AIM-deficient (AIM(-/-)) mice, the increase of multiple IgG autoantibodies observed in obese wild-type mice was abrogated. Thus, the AIM-IgM association plays a critical role in the obesity-associated autoimmune process.

The PTPN22 1858T allele but not variants in the proximal promoter region ofIL-21gene is associated with the susceptibility to type 1 diabetes and the presence of autoantibodies in a Brazilian cohort

Interleukin (IL)-21 and protein tyrosine phosphatase non-receptor 22 (PTPN22) regulate lymphocyte function and have been implicated in the pathogenesis of autoimmune diabetes. We sequenced the proximal promoter of the IL-21 gene for the first time and analysed the PTPN22 1858T polymorphism in type 1A diabetes (T1AD) patients and healthy controls (HC). We correlated the frequencies of islet and extra-pancreatic autoantibodies with genotypes from both loci. The case series comprised 612 T1AD patients and 792 HC. Genotyping of PTPN22 C1858T was performed on 434 T1AD patients and 689 HC. The -448 to +83 base pairs (bp) region of the IL-21 gene was sequenced in 309 Brazilian T1AD and 189 HC subjects. We also evaluated human leucocyte antigen (HLA) DR3/DR4 alleles. The frequencies of glutamic acid decarboxylase (GAD65), tyrosine phosphatase-like protein (IA)-2, anti-nuclear antibody (ANA), thyroid peroxidase (TPO), thyroglobulin (TG), thyrotrophin receptor autoantibody (TRAb), anti-smooth muscle (ASM) and 21-hydroxylase (21-OH) autoantibodies were higher in T1AD patients than in HC. The PTPN22 1858T allele was associated with an increased risk for developing T1AD [odds ratio (OR) = 1·94; P < 0·001], particularly in patients of European ancestry, and with a higher frequency of GAD65 and TG autoantibodies. HLA-DR3/DR4 alleles predominated in T1AD patients. A heterozygous allelic IL-21 gene variant (g.-241 T > A) was found in only one patient. In conclusion, only PTPN22 C1858T polymorphism and HLA-DR3 and/or DR4 alleles, but not allelic variants in the 5'-proximal region of the IL-21 gene were associated with T1AD risk. Patients with T1AD had increased frequencies of anti-islet-cell, anti-thyroid, anti-nuclear, anti-smooth muscle and anti-21-OH autoantibodies. The C1858T PTPN22 polymorphism was also associated with a higher frequency of GAD65 and TG autoantibodies.

Prevalence of zinc transporter 8 antibodies in gestational diabetes mellitus

Gestational diabetes mellitus affects approximately 7% of all pregnant women. Some of these women develop autoantibodies that are generally characteristic of Type 1 diabetes. Autoantibodies targeting glutamic acid decarboxylase and tyrosine phosphatase-like protein are the most frequently reported. A recently identified autoantigen in Type 1 diabetes is zinc transporter 8. Some reports suggest that the frequency of zinc transporter 8 antibodies is as high as glutamic acid decarboxylase antibodies in Type 1 diabetes and thus a good diagnostic marker for autoimmune diabetes. There are currently no reports of zinc transporter 8 antibodies in gestational diabetes. The aim of this pilot study was to investigate the frequency of zinc transporter 8 antibodies in patients at clinical onset of gestational diabetes mellitus. Subjects included in this pilot study were all diagnosed with gestational diabetes at Skåne University Hospital, Lund, Sweden, 2009-2010 (n = 193). Sera samples were analysed for antibodies using a commercial enzyme-linked immunosorbent assay according to the manufacturers' instructions. We found that 19/193 patients with gestational diabetes, diagnosed in 2009-2010, were positive for at least one autoantibody. Glutamic acid decarboxylase was the most common single autoantibody (52.6%; 10/19), followed by zinc transporter 8 (21.1%; 4/19) and tyrosine phosphatase-like protein (15.8%; 3/19). Combinations of two or more antibodies were rare (10.5%; 2/19). In this study, we found that zinc transporter 8 added 2.1% (4/193) of autoantibody positivity in women with gestational diabetes who were negative for glutamic acid decarboxylase and tyrosine phosphatase-like protein antibodies. Glutamic acid decarboxylase was still the most prevalent autoantibody in gestational diabetes, but, as zinc transporter 8 was present even in the absence of glutamic acid decarboxylase, this autoantibody could be an important independent marker of autoimmunity in gestational diabetes.

Prevalence of ZnT8 antibody in relation to phenotype and SLC30A8 polymorphism in adult autoimmune diabetes. Results from the HUNT study, Norway

Zinc transporter 8 (ZnT8), a product of the SLC30A8 gene, is a target-antigen in autoimmune diabetes. Associations between ZnT8 antibody (ZnT8A), phenotype and the genetic variant rs13266634 in the SLC30A8 gene have primarily been studied in patients with young-onset diabetes. We explored such associations in adult-onset autoimmune diabetes identified from the all-population based Nord-Trøndelag health Study (HUNT) ZnT8A (assayed by a fusion probe of C-terminal Arg325 and Trp325), and antibodies against glutamic decarboxylase (GADA) and tyrosine phosphatase-like protein insulinoma antigen-2 (IA-2A) were analysed in 266 subjects classified as having adult-onset autoimmune diabetes ( ≥ 25 years of age at diagnosis). Of these, 161 subjects fulfilled the criteria of latent autoimmune diabetes in adults (LADA), whereas 105 subjects were termed “classical” type 1 diabetes. Ten out of 161 LADA (6.2%) and 23 out of 105 adult-onset “classical” type 1 diabetic patients (22%) were ZnT8A positive. Adult-onset diabetic subjects positive both for GADA and IA-2A (n = 17), had lower waist circumference (p = 0.024) and higher fasting glucose levels (p = 0.023) than those positive both for GADA and ZnT8A (n = 13). Genotyping results of rs13266634 (available in 178 adult-onset diabetic subjects), showed a tendency for association between ZnT8A positivity and the TT- and CC genotypes of SNP rs13266634 (p = 0.101) using the standard cut-off level of 0.06ai, and a significant association at a lower cut-off level of 0.01ai (p = 0.005). We conclude that ZnT8A positivity in a population of adult-onset autoimmune diabetes is a less strong marker of autoimmunity than IA-2A. Further, positivity could be influenced by polymorphism of the SLC30A8 gene.