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1.  Risk of Pediatric Celiac Disease According to HLA Haplotype and Country 
The presence of HLA haplotype DR3–DQ2 or DR4–DQ8 is associated with an increased risk of celiac disease. In addition, nearly all children with celiac disease have serum antibodies against tissue transglutaminase (tTG).
We studied 6403 children with HLA haplotype DR3–DQ2 or DR4–DQ8 prospectively from birth in the United States, Finland, Germany, and Sweden. The primary end point was the development of celiac disease autoimmunity, which was defined as the presence of tTG antibodies on two consecutive tests at least 3 months apart. The secondary end point was the development of celiac disease, which was defined for the purpose of this study as either a diagnosis on biopsy or persistently high levels of tTG antibodies.
The median follow-up was 60 months (interquartile range, 46 to 77). Celiac disease autoimmunity developed in 786 children (12%). Of the 350 children who underwent biopsy, 291 had confirmed celiac disease; an additional 21 children who did not undergo biopsy had persistently high levels of tTG antibodies. The risks of celiac disease autoimmunity and celiac disease by the age of 5 years were 11% and 3%, respectively, among children with a single DR3–DQ2 haplotype, and 26% and 11%, respectively, among those with two copies (DR3–DQ2 homozygosity). In the adjusted model, the hazard ratios for celiac disease autoimmunity were 2.09 (95% confidence interval [CI], 1.70 to 2.56) among heterozygotes and 5.70 (95% CI, 4.66 to 6.97) among homozygotes, as compared with children who had the lowest-risk genotypes (DR4–DQ8 heterozygotes or homozygotes). Residence in Sweden was also independently associated with an increased risk of celiac disease autoimmunity (hazard ratio, 1.90; 95% CI, 1.61 to 2.25).
Children with the HLA haplotype DR3–DQ2, especially homozygotes, were found to be at high risk for celiac disease autoimmunity and celiac disease early in childhood. The higher risk in Sweden than in other countries highlights the importance of studying environmental factors associated with celiac disease. (Funded by the National Institute of Diabetes and Digestive and Kidney Diseases and others.)
PMCID: PMC4163840  PMID: 24988556
2.  A type I interferon transcriptional signature precedes autoimmunity in children genetically at-risk of type 1 diabetes 
Diabetes  2014;63(7):2538-2550.
Diagnosis of the autoimmune disease type 1 diabetes (T1D) is preceded by the appearance of circulating autoantibodies to pancreatic islets. However, almost nothing is known about events leading to this islet autoimmunity. Previous epidemiological and genetic data have associated viral infections and anti-viral type I interferon (IFN) immune response genes with T1D. Here, we first used DNA microarray analysis to identify IFN-β inducible genes in vitro and then used this set of genes to define an IFN-inducible transcriptional signature in peripheral blood mononuclear cells from a group of active systemic lupus erythematosus patients (N=25). Using this predefined set of 225 IFN signature genes, we investigated expression of the signature in cohorts of healthy controls (N=87), T1D patients (N=64) and a large longitudinal birth cohort of children genetically predisposed to T1D (N=109; 454 microarrayed samples). Expression of the IFN signature was increased in genetically-predisposed children prior to the development of autoantibodies (P=0.0012), but not in established T1D patients. Upregulation of IFN-inducible genes was transient, temporally associated with a recent history of upper respiratory tract infections (P=0.0064) and marked by increased expression of SIGLEC-1 (CD169), a lectin-like receptor expressed on CD14+ monocytes. DNA variation in IFN-inducible genes altered T1D risk (P=0.007), as exemplified by IFIH1, one of the genes in our IFN signature and for which increased expression is a known disease risk factor. These findings identify transient increased expression of type I IFN genes in pre-clinical diabetes as a risk factor for autoimmunity in children with a genetic predisposition to T1D.
PMCID: PMC4066333  PMID: 24561305
3.  Concentration and Activity of the Soluble Form of the Interleukin-7 Receptor α in Type 1 Diabetes Identifies an Interplay Between Hyperglycemia and Immune Function 
Diabetes  2013;62(7):2500-2508.
Soluble interleukin-7 (IL-7) receptor α (sCD127) is implicated in the pathogenesis of autoimmune diseases. We show that serum sCD127 concentrations are increased at the onset of type 1 diabetes (T1D; n = 390) as compared with concentrations in age-matched islet autoantibody–negative first-degree relatives of patients (n = 392; P = 0.00001). sCD127 concentration in patients was influenced by islet autoantibody status (P = 0.003) and genotype of the rs6897932 single nucleotide polymorphism within the IL-7RA gene (P = 0.006). Release of sCD127 in vitro was strongly upregulated by activation of T lymphocytes and affected by exposure to cytokines. sCD127 bound IL-7 and was antagonistic to IL-7 signaling and IL-7–mediated T-cell proliferation, suggesting a regulatory feedback mechanism on T-cell expansion. Remarkably, high glucose led to a glycated form of sCD127 that was ineffective as an IL-7 antagonist. The finding of glycated sCD127 in the circulation of patients at onset of T1D suggested that physiological regulation of IL-7–mediated T-cell survival and expansion by sCD127 may be compromised in T1D. The findings indicate that genetic, immunologic, and metabolic factors contribute to a dysregulation of the IL-7/IL-7 receptor pathway in T1D and identify a novel hyperglycemia-mediated interference of immune regulatory networks.
PMCID: PMC3712069  PMID: 23454692
4.  Activation of Islet Autoreactive Naïve T Cells in Infants Is Influenced by Homeostatic Mechanisms and Antigen-Presenting Capacity 
Diabetes  2013;62(6):2059-2066.
Islet autoimmunity precedes type 1 diabetes onset. We previously found that islet autoimmunity rarely starts before 6 months of age but reaches its highest incidence already at ∼1 year of age. We now examine whether homeostatic expansion and immune competence changes seen in a maturating immune system may account for this marked variation in islet autoimmunity risk in the first year of life. We found naïve proinsulin- and GAD65-responsive T cells in cord blood (CB) of healthy newborns, with highest responses observed in children with type 1 diabetes-susceptible HLA-DRB1/DQB1 genotypes. Homeostatic expansion characteristics with increased IL-7 concentrations and enhanced T-cell responsiveness to IL-7 were observed throughout the first year of life. However, the ability of antigen-presenting cells to activate naïve T cells was compromised at birth, and CB monocytes had low surface expression of CD40 and HLA class II. In contrast, antigen presentation and expression of these molecules had reached competent adult levels by the high incidence age of 8 months. We propose that temporal changes in islet autoimmunity seroconversion in infants are a consequence of the changing balance between homeostatic drive and antigen presentation competence. These findings are relevant for early prevention of type 1 diabetes.
PMCID: PMC3661654  PMID: 23349478
5.  Alloantibody and Autoantibody Monitoring Predicts Islet Transplantation Outcome in Human Type 1 Diabetes 
Diabetes  2013;62(5):1656-1664.
Long-term clinical outcome of islet transplantation is hampered by the rejection and recurrence of autoimmunity. Accurate monitoring may allow for early detection and treatment of these potentially compromising immune events. Islet transplant outcome was analyzed in 59 consecutive pancreatic islet recipients in whom baseline and de novo posttransplant autoantibodies (GAD antibody, insulinoma-associated protein 2 antigen, zinc transporter type 8 antigen) and donor-specific alloantibodies (DSA) were quantified. Thirty-nine recipients (66%) showed DSA or autoantibody increases (de novo expression or titer increase) after islet transplantation. Recipients who had a posttransplant antibody increase showed similar initial performance but significantly lower graft survival than patients without an increase (islet autoantibodies P < 0.001, DSA P < 0.001). Posttransplant DSA or autoantibody increases were associated with HLA-DR mismatches (P = 0.008), induction with antithymocyte globulin (P = 0.0001), and pretransplant panel reactive alloantibody >15% in either class I or class II (P = 0.024) as independent risk factors and with rapamycin as protective (P = 0.006) against antibody increases. DSA or autoantibody increases after islet transplantation are important prognostic markers, and their identification could potentially lead to improved islet cell transplant outcomes.
PMCID: PMC3636624  PMID: 23274902
6.  Immunotherapy in Type 1 Diabetes: A Shorter but More Winding Road? 
Diabetes  2012;61(9):2214-2215.
PMCID: PMC3425430  PMID: 22923646
7.  Cesarean Section and Interferon-Induced Helicase Gene Polymorphisms Combine to Increase Childhood Type 1 Diabetes Risk 
Diabetes  2011;60(12):3300-3306.
The incidence of type 1 diabetes is increasing. Delivery by cesarean section is also more prevalent, and it is suggested that cesarean section is associated with type 1 diabetes risk. We examine associations between cesarean delivery, islet autoimmunity and type 1 diabetes, and genes involved in type 1 diabetes susceptibility.
Cesarean section was examined as a risk factor in 1,650 children born to a parent with type 1 diabetes and followed from birth for the development of islet autoantibodies and type 1 diabetes.
Children delivered by cesarean section (n = 495) had more than twofold higher risk for type 1 diabetes than children born by vaginal delivery (hazard ratio [HR] 2.5; 95% CI 1.4–4.3; P = 0.001). Cesarean section did not increase the risk for islet autoantibodies (P = 0.6) but was associated with a faster progression to diabetes after the appearance of autoimmunity (P = 0.015). Cesarean section–associated risk was independent of potential confounder variables (adjusted HR 2.7;1.5–5.0; P = 0.001) and observed in children with and without high-risk HLA genotypes. Interestingly, cesarean section appeared to interact with immune response genes, including CD25 and in particular the interferon-induced helicase 1 gene, where increased risk for type 1 diabetes was only seen in children who were delivered by cesarean section and had type 1 diabetes–susceptible IFIH1 genotypes (12-year risk, 9.1 vs. <3% for all other combinations; P < 0.0001).
These findings suggest that type 1 diabetes risk modification by cesarean section may be linked to viral responses in the preclinical autoantibody-positive disease phase.
PMCID: PMC3219940  PMID: 22110093
8.  Expansion of Th17 Cells and Functional Defects in T Regulatory Cells Are Key Features of the Pancreatic Lymph Nodes in Patients With Type 1 Diabetes 
Diabetes  2011;60(11):2903-2913.
Autoimmune diseases, including type 1 diabetes, are thought to have a Th17-cell bias and/or a T-regulatory cell (Treg) defect. Understanding whether this is a hallmark of patients with type 1 diabetes is a crucial question that is still unsolved, largely due to the difficulties of accessing tissues targeted by the disease.
We phenotypically and functionally characterized Th17 cells and Tregs residing in the pancreatic-draining lymph nodes (PLNs) of 19 patients with type 1 diabetes and 63 nondiabetic donors and those circulating in the peripheral blood of 14 type 1 diabetic patients and 11 healthy subjects.
We found upregulation of Th17 immunity and functional defects in CD4+CD25bright Tregs in the PLNs of type 1 diabetic subjects but not in their peripheral blood. In addition, the proinsulin-specific Treg-mediated control was altered in the PLNs of diabetic patients. The dysfunctional Tregs isolated from diabetic subjects did not contain contaminant effector T cells and were all epigenetically imprinted to be suppressive, as defined by analysis of the Treg-specific demethylated region within the forkhead box P3 (FOXP3) locus.
These data provide evidence for an unbalanced immune status in the PLNs of type 1 diabetic subjects, and treatments restoring the immune homeostasis in the target organ of these patients represent a potential therapeutic strategy.
PMCID: PMC3198077  PMID: 21896932
9.  Age- and Islet Autoimmunity–Associated Differences in Amino Acid and Lipid Metabolites in Children at Risk for Type 1 Diabetes 
Diabetes  2011;60(11):2740-2747.
Islet autoimmunity precedes type 1 diabetes and often initiates in childhood. Phenotypic variation in islet autoimmunity relative to the age of its development suggests heterogeneous mechanisms of autoimmune activation. To support this notion, we examined whether serum metabolite profiles differ between children with respect to islet autoantibody status and the age of islet autoantibody development.
The study analyzed 29 metabolites of amino acid metabolism and 511 lipids assigned to 12 lipid clusters in children, with a type 1 diabetic parent, who first developed autoantibodies at age 2 years or younger (n = 13), at age 8 years or older (n = 22), or remained autoantibody-negative, and were matched for age, date of birth, and HLA genotypes (n = 35). Ultraperformance liquid chromatography and mass spectroscopy were used to measure metabolites and lipids quantitatively in the first autoantibody-positive and matched autoantibody-negative serum samples and in a second sample after 1 year of follow-up.
Differences in the metabolite profiles were observed relative to age and islet autoantibody status. Independent of age-related differences, autoantibody-positive children had higher levels of odd-chain triglycerides and polyunsaturated fatty acid–containing phospholipids than autoantibody-negative children and independent of age at first autoantibody appearance (P < 0.0001). Consistent with our hypothesis, children who developed autoantibodies by age 2 years had twofold lower concentration of methionine compared with those who developed autoantibodies in late childhood or remained autoantibody-negative (P < 0.0001).
Distinct metabolic profiles are associated with age and islet autoimmunity. Pathways that use methionine are potentially relevant for developing islet autoantibodies in early infancy.
PMCID: PMC3198092  PMID: 22025777
10.  CXCR1/2 inhibition enhances pancreatic islet survival after transplantation 
The Journal of Clinical Investigation  2012;122(10):3647-3651.
Although long considered a promising treatment option for type 1 diabetes, pancreatic islet cell transformation has been hindered by immune system rejection of engrafted tissue. The identification of pathways that regulate post-transplant detrimental inflammatory events would improve management and outcome of transplanted patients. Here, we found that CXCR1/2 chemokine receptors and their ligands are crucial negative determinants for islet survival after transplantation. Pancreatic islets released abundant CXCR1/2 ligands (CXCL1 and CXCL8). Accordingly, intrahepatic CXCL1 and circulating CXCL1 and CXCL8 were strongly induced shortly after islet infusion. Genetic and pharmacological blockade of the CXCL1-CXCR1/2 axis in mice improved intrahepatic islet engraftment and reduced intrahepatic recruitment of polymorphonuclear leukocytes and NKT cells after islet infusion. In humans, the CXCR1/2 allosteric inhibitor reparixin improved outcome in a phase 2 randomized, open-label pilot study with a single infusion of allogeneic islets. These findings indicate that the CXCR1/2-mediated pathway is a regulator of islet damage and should be a target for intervention to improve the efficacy of transplantation.
PMCID: PMC3461913  PMID: 22996693
11.  Accelerated progression from islet autoimmunity to diabetes is causing the escalating incidence of type 1 diabetes in young children 
Journal of autoimmunity  2011;37(1):3-7.
The incidence of type 1 diabetes is rising worldwide, particularly in young children. Since type 1 diabetes is preceded by autoimmunity to islet antigens, there must be a consequent increase in the incidence of islet autoimmunity in young children or a more rapid rate of progression to diabetes once islet autoimmunity initiates. This study was to determine whether the incidence of islet autoimmunity or the rate of progression from autoimmunity to diabetes onset has changed over a 20-year period in children genetically predisposed to type 1 diabetes. Between 1989 and 2010, children who were first-degree relatives of patients with type 1 diabetes and who were born in Germany were prospectively followed from birth without intervention. A total of 324 children (BABYDIAB study) born between 1989 and 2000 and 216 children (TEDDY study) born between 2004 and 2010 with matched HLA genotypes were recruited before age 3 months and included for analysis. Children were followed for the development of autoantibodies to insulin, GAD, and IA-2, and for progression to diabetes. The cumulative frequency of diabetes by age 4 years was 2.5% (95% CI 0.8–4.2%) in BABYDIAB children and 6.2% (95% CI 2.3–10.1%) in TEDDY children (p=0.03). The cumulative frequency of islet autoantibodies by age 4 years was similar in the children from both studies (11.3% vs 13.9%). Progression to diabetes from the development of islet autoantibodies was markedly increased in autoantibody-positive children from the more recently recruited TEDDY cohort (50% progression within 85.2 months for BABYDIAB children vs 9.6 months for TEDDY children; p=0.009), also if children were further selected on the basis of high-risk HLA genotypes or the development of autoantibodies to multiple islet antigens (p=0.01). The findings suggest that recent increasing incidence of type 1 diabetes in young children could be due to weakening of mechanisms that normally regulate autoimmune destruction of islet beta cells.
PMCID: PMC3110538  PMID: 21376535
type 1 diabetes; islet autoimmunity; incidence; progression
12.  Primary Dietary Intervention Study to Reduce the Risk of Islet Autoimmunity in Children at Increased Risk for Type 1 Diabetes 
Diabetes Care  2011;34(6):1301-1305.
To determine whether delaying the introduction of gluten in infants with a genetic risk of islet autoimmunity is feasible, safe, and may reduce the risk of type 1 diabetes–associated islet autoimmunity.
A total of 150 infants with a first-degree family history of type 1 diabetes and a risk HLA genotype were randomly assigned to a first gluten exposure at age 6 months (control group) or 12 months (late-exposure group) and were followed 3 monthly until the age of 3 years and yearly thereafter for safety (for growth and autoantibodies to transglutaminase C [TGCAs]), islet autoantibodies to insulin, GAD, insulinoma-associated protein 2, and type 1 diabetes.
Adherence to the dietary-intervention protocol was reported from 70% of families. During the first 3 years, weight and height were similar in children in the control and late-exposure groups, as was the probability of developing TGCAs (14 vs. 4%; P = 0.1). Eleven children in the control group and 13 children in the late-exposure group developed islet autoantibodies (3-year risk: 12 vs. 13%; P = 0.6). Seven children developed diabetes, including four in the late-exposure group. No significant differences were observed when children were analyzed as per protocol on the basis of the reported first gluten exposure of the children.
Delaying gluten exposure until the age of 12 months is safe but does not substantially reduce the risk for islet autoimmunity in genetically at-risk children.
PMCID: PMC3114350  PMID: 21515839
13.  An Interferon-Induced Helicase (IFIH1) Gene Polymorphism Associates With Different Rates of Progression From Autoimmunity to Type 1 Diabetes 
Diabetes  2011;60(2):685-690.
Genome-wide association studies have identified gene regions associated with the development of type 1 diabetes. The aim of this study was to determine whether these associations are with the development of autoimmunity and/or progression to diabetes.
Children (n = 1,650) of parents with type 1 diabetes were prospectively followed from birth (median follow-up 10.20 years) for the development of islet autoantibodies, thyroid peroxidase antibodies, tissue transglutaminase antibodies, and diabetes. Genotyping for single-nucleotide polymorphisms of the PTPN22, ERBB3, PTPN2, KIAA0350, CD25, and IFIH1 genes was performed using the MassARRAY system with iPLEX chemistry.
Islet autoantibodies developed in 137 children and diabetes developed in 47 children. Type 1 diabetes risk was associated with the IFIH1 rs2111485 single-nucleotide polymorphism (hazard ratio 2.08; 95% CI 1.16–3.74; P = 0.014). None of the other genes were significantly associated with diabetes development in this cohort. IFIH1 genotypes did not associate with the development of islet autoantibodies (P = 0.80) or autoantibodies against thyroid peroxidase (P = 0.55) and tissue transglutaminase (P = 0.66). Islet autoantibody–positive children with the IFIH1 rs2111485 GG genotype had a faster progression to diabetes (31% within 5 years) than children with the type 1 diabetes protective GA or AA genotypes (11% within 5 years; P = 0.006).
The findings indicate that IFIH1 genotypes influence progression from autoimmunity to diabetes development, consistent with the notion that protective genotypes downregulate responses to environmental insults after initiation of autoimmunity.
PMCID: PMC3028371  PMID: 21270278
14.  BMI at Age 8 Years Is Influenced by the Type 2 Diabetes Susceptibility Genes HHEX-IDE and CDKAL1 
Diabetes  2010;59(8):2063-2067.
To determine whether HHEX-IDE and CDKAL1 genes, which are associated with birth weight and susceptibility to type 2 diabetes, continue to influence growth during childhood.
BMI, weight, and height at age 8 years expressed as age- and sex-corrected standard deviation scores (SDS) against national reference data and single-nucleotide polymorphism genotyping of HHEX-IDE and CDKAL1 loci were analyzed in 646 prospectively followed children in the German BABYDIAB cohort. All children were singleton full-term births; 386 had mothers with type 1 diabetes, and 260 had fathers with type 1 diabetes and a nondiabetic mother.
Type 2 diabetes risk alleles at the HHEX-IDE locus were associated with reduced BMI-SDS at age 8 years (0.17 SDS per allele; P = 0.004). After stratification for birth weight, both HHEX-IDE and CDKAL1 risk alleles were associated with reduced BMI-SDS (0.45 SDS, P = 0.0002; 0.52 SDS, P = 0.0001) and weight-SDS (0.22 SDS, P = 0.04; 0.56 SDS, P = 0.0002) in children born large for gestational age (>90th percentile) but not children born small or appropriate for gestational age. Within children born large for gestational age, BMI and weight decreased with each additional type 2 diabetes risk allele (∼ −2 kg per allele; >8 kg overall). Findings were consistent in children of mothers with type 1 diabetes (P < 0.0001) and children of nondiabetic mothers (P = 0.008).
The type 2 diabetes susceptibility alleles at HHEX-IDE and CDKAL1 loci are associated with low BMI at age 8 years in children who were born large for gestational age.
PMCID: PMC2911059  PMID: 20460429
15.  The Use of Biomaterials in Islet Transplantation 
Current Diabetes Reports  2011;11(5):434-444.
Pancreatic islet transplantation is a therapeutic option to replace destroyed β cells in autoimmune diabetes. Islets are transplanted into the liver via the portal vein; however, inflammation, the required immunosuppression, and lack of vasculature decrease early islet viability and function. Therefore, the use of accessory therapy and biomaterials to protect islets and improve islet function has definite therapeutic potential. Here we review the application of niche accessory cells and factors, as well as the use of biomaterials as carriers or capsules, for pancreatic islet transplantation.
PMCID: PMC3167046  PMID: 21748257
Type 1 diabetes; Islet transplantation; Islet niche; Biomaterial; Encapsulation
16.  No Effect of the 1α,25-Dihydroxyvitamin D3 on β-Cell Residual Function and Insulin Requirement in Adults With New-Onset Type 1 Diabetes 
Diabetes Care  2010;33(7):1443-1448.
To determine whether daily intake of 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3] is safe and improves β-cell function in patients with recently diagnosed type 1 diabetes.
Safety was assessed in an open study of 25 patients aged 18–39 years with recent-onset type 1 diabetes who received 0.25 μg 1,25(OH)2D3 daily for 9 months. An additional 40 patients were randomly assigned to 0.25 μg 1,25(OH)2D3 or placebo daily for 9 months and followed for a total of 18 months for safety, β-cell function, insulin requirement, and glycemic control.
Safety assessment showed values in the normal range in nearly all patients, regardless of whether they received 1,25(OH)2D3 or placebo. No differences in AUC C-peptide, peak C-peptide, and fasting C-peptide after a mixed-meal tolerance test between the treatment and placebo groups were observed at 9 and 18 months after study entry, with ∼40% loss for each parameter over the 18-month period. A1C and daily insulin requirement were similar between treatment and placebo groups throughout the study follow-up period.
Treatment with 1,25(OH)2D3 at a daily dose of 0.25 μg was safe but did not reduce loss of β-cell function.
PMCID: PMC2890336  PMID: 20357369
17.  Zinc Transporter 8 Antibodies Complement GAD and IA-2 Antibodies in the Identification and Characterization of Adult-Onset Autoimmune Diabetes 
Diabetes Care  2009;33(1):104-108.
Zinc transporter 8 (ZnT8) is an islet β-cell secretory granule membrane protein recently identified as an autoantibody antigen in type 1 diabetes. The aim of this study was to determine the prevalence and role of antibodies to ZnT8 (ZnT8As) in adult-onset diabetes.
ZnT8As were measured by a radioimmunoprecipitation assay using recombinant ZnT8 COOH-terminal or NH2-terminal proteins in 193 patients with adult-onset autoimmune diabetes having antibodies to either GAD (GADAs) or IA-2 (IA-2As) and in 1,056 antibody-negative patients with type 2 diabetes from the Non Insulin Requiring Autoimmune Diabetes (NIRAD) study.
ZnT8As-COOH were detected in 18.6% patients with autoimmune diabetes and 1.4% with type 2 diabetes. ZnT8As-NH2 were rare. ZnT8As were associated with younger age and a high GADA titer. The use of GADAs, IA-2As, and ZnT8As in combination allowed a stratification of clinical phenotype, with younger age of onset of diabetes and characteristics of more severe insulin deficiency (higher fasting glucose and A1C, lower BMI, total cholesterol, and triglycerides) in patients with all three markers, with progressive attenuation in patients with two, one, and no antibodies (all Ptrend < 0.001). Autoantibody titers, association with high-risk HLA genotypes, and prevalence of thyroid peroxidase antibodies followed the same trend (all P < 0.001).
ZnT8As are detectable in a proportion of patients with adult-onset autoimmune diabetes and seem to be a valuable marker to differentiate clinical phenotypes.
PMCID: PMC2797953  PMID: 19808923
18.  Azide and Tween-20 reduce binding to autoantibody epitopes of islet antigen-2; implications for assay performance and reproducibility 
Journal of immunological methods  2009;351(1-2):75-79.
Autoantibodies to islet antigen 2 (IA-2A) are important markers for predicting diabetes in children and young adults. Harmonization of IA-2A assay measurement is essential if results from different laboratories are to be compared. We investigated whether sodium azide, a bacteriostatic agent added to some assays, could affect IA-2A binding and thereby contribute to differences in IA-2A measurement between laboratories. Addition of 0.1 % azide to assay buffer was found to reduce median IA-2A binding of 18 selected sera from IA-2A positive patients with type 1 diabetes and their relatives by 41 % (range, 78 to -33 %, p<0.001). The effect on binding was epitope specific; median IA-2A binding by 14 sera with antibodies to the protein tyrosine phosphatase region of IA-2 was reduced by 48% (range, 11 to 78 %, p<0.001), while binding by 4 sera with antibodies specific to only the juxtamembrane region of IA-2 showed no change (median increase 16 % (range 6 to 33 %, p=0.125). When the Tween-20 concentration was reduced from 1 % to 0.15 % the median reduction in IA-2A binding with azide by the 18 sera was only 10 % (range, -12 to 41 %, p<0.001). Tween-20 also exerted an independent effect, since median IA-2A binding increased by 23 % (range 3 % to 86 %, p<0.001) when Tween-20 concentration was reduced from 1 % to 0.15 % in the absence of azide. We conclude that common assay reagents such as azide and Tween-20 can strongly influence IA-2A binding in an epitope-related manner, and their use may explain some of the differences between laboratories in IA-2A measurement.
PMCID: PMC2830747  PMID: 19837075
Islet autoantibody assay; epitope; protein tyrosine phosphatase; Tween-20; azide; type 1 diabetes
19.  A Report on the International Transglutaminase Autoantibody Workshop for Celiac Disease 
Measurement of transglutaminase autoantibodies (TGAA) is considered to be the most efficient single serologic test for celiac disease (CD) by the American Gastroenterological Association Institute. We hypothesized that a large international collaborative effort toward improving and standardizing TGAA measurement is both feasible and necessary. The primary aim of this workshop is to compare TGAA assays among various research and clinical laboratories and examine assay concordance and improve (and eventually standardize) the TGAA assay.
A total of 20 laboratories (5 commercial laboratories, 15 research and clinical laboratories) participated that included enzyme-linked immunosorbent assay (ELISA) and radiobinding assays. A total of 150 serum samples were distributed to each laboratory, with each laboratory receiving an equal aliquot that was coded and blinded, composed of 100 healthy control sera and 50 CD sera.
Laboratory sensitivity ranged from 69% to 93% and specificity ranged from 96% to 100%. By receiver operator characteristic analysis, the area under the curve (C index) ranged from 0.9488 to 0.9904. When analyzing for linear correlation, r-squared was as high as 0.8882 but as low as 0.4244 for the celiac samples between different laboratories performing ELISA.
This transglutaminase autoantibody workshop allows for larger-scale international participation for the purposes of improving and eventually standardizing the TGAA assay with subsequent workshops.
PMCID: PMC2904747  PMID: 19098864
20.  Co-Graft of Allogeneic Immune Regulatory Neural Stem Cells (NPC) and Pancreatic Islets Mediates Tolerance, while Inducing NPC-Derived Tumors in Mice 
PLoS ONE  2010;5(4):e10357.
Data available on the immunomodulatory properties of neural stem/precursor cells (NPC) support their possible use as modulators for immune-mediated process. The aim of this study was to define whether NPC administered in combination with pancreatic islets prevents rejection in a fully mismatched allograft model.
Methodology/Principal Finding
Diabetic Balb/c mice were co-transplanted under the kidney capsule with pancreatic islets and GFP+ NPC from fully mismatched C57BL/6 mice. The following 4 groups of recipients were used: mice receiving islets alone; mice receiving islets alone and treated with standard immunosuppression (IL-2Rα chain mAbs + FK506 + Rapamycin); mice receiving a mixed islet/NPC graft under the same kidney capsule (Co-NPC-Tx); mice receiving the islet graft under the left kidney capsule and the NPC graft under the right kidney capsule (NPC-Tx). Our results demonstrate that only the co-transplantation and co-localization of NPC and islets (Co-NPC-Tx) induce stable long-term graft function in the absence of immunosuppression. This condition is associated with an expansion of CD4+CD25+FoxP3+ T regulatory cells in the spleen. Unfortunately, stable graft function was accompanied by constant and reproducible development of NPC-derived cancer mainly sustained by insulin secretion.
These data demonstrate that the use of NPC in combination with islets prevents graft rejection in a fully mismatched model. However, the development of NPC-derived cancer raises serious doubts about the safety of using adult stem cells in combination with insulin-producing cells outside the original microenvironment.
PMCID: PMC2860511  PMID: 20436918
21.  Rapamycin Monotherapy in Patients With Type 1 Diabetes Modifies CD4+CD25+FOXP3+ Regulatory T-Cells 
Diabetes  2008;57(9):2341-2347.
OBJECTIVE—Rapamycin is an immunosuppressive drug currently used to prevent graft rejection in humans, which is considered permissive for tolerance induction. Rapamycin allows expansion of both murine and human naturally occurring CD4+CD25+FOXP3+ T regulatory cells (nTregs), which are pivotal for the induction and maintenance of peripheral tolerance. Preclinical murine models have shown that rapamycin enhances nTreg proliferation and regulatory function also in vivo. Objective of this study was to assess whether rapamycin has in vivo effects on human nTregs.
RESEARCH DESIGN AND METHODS—nTreg numbers and function were examined in a unique set of patients with type 1 diabetes who underwent rapamycin monotherapy before islet transplantation.
RESULTS—We found that rapamycin monotherapy did not alter the frequency and functional features, namely proliferation and cytokine production, of circulating nTregs. However, nTregs isolated from type 1 diabetic patients under rapamycin treatment had an increased capability to suppress proliferation of CD4+CD25− effector T-cells compared with that before treatment.
CONCLUSIONS—These findings demonstrate that rapamycin directly affects human nTreg function in vivo, which consists of refitting their suppressive activity, whereas it does not directly change effector T-cell function.
PMCID: PMC2518485  PMID: 18559659
22.  Influence of Early Nutritional Components on the Development of Murine Autoimmune Diabetes 
Annals of Nutrition & Metabolism  2009;54(3):208-217.
Infant diet is suggested to modify autoimmune diabetes risk. The aim of this study was to determine whether infant food components affect diabetes development in the nonobese autoimmune diabetes (NOD) mouse.
A basal low-diabetogenic diet was identified by feeding litter-matched female NOD mice standardized diets with and without casein and wheat proteins after weaning. In subsequent trials, basal diet with supplements of wheat (5, 10 and 30%), gluten, wheat globulin/albumin, corn (5%), potato (5%), apple (5%) or carrot (5%) was fed to litter-matched female NOD mice after weaning. Mice were followed for diabetes development and insulin autoantibodies.
A casein- and wheat-free diet was associated with the lowest rate of diabetes development (37% by age 25 weeks). Increased diabetes rates were observed when the basal diet was supplemented with 5% wheat (71% by age 25 weeks; p = 0.023) and 5% corn (57% by age 25 weeks; p = 0.05). Increasing wheat concentrations returned diabetes development to that in basal diet-fed mice. Other food supplements had no or minimal effects on diabetes development.
Early supplementation of a basal low-diabetogenic diet with low concentrations of the cereals wheat or corn is associated with a moderate increase in the rate of diabetes. Removal of cereals, however, does not abrogate diabetes development in NOD mice.
PMCID: PMC2814014  PMID: 19478481
Type 1 diabetes; Autoimmunity; NOD mice; Diet; Prevention; Gluten; Casein
23.  Islet transplantation in patients with autoimmune diabetes induces homeostatic cytokines that expand autoreactive memory T cells 
The Journal of Clinical Investigation  2008;118(5):1806-1814.
Successful transplantation requires the prevention of allograft rejection and, in the case of transplantation to treat autoimmune disease, the suppression of autoimmune responses. The standard immunosuppressive treatment regimen given to patients with autoimmune type 1 diabetes who have received an islet transplant results in the loss of T cells. In many other situations, the immune system responds to T cell loss through cytokine-dependant homeostatic proliferation of any remaining T cells. Here we show that T cell loss after islet transplantation in patients with autoimmune type 1 diabetes was associated with both increased serum concentrations of IL-7 and IL-15 and in vivo proliferation of memory CD45RO+ T cells, highly enriched in autoreactive glutamic acid decarboxylase 65–specific T cell clones. Immunosuppression with FK506 and rapamycin after transplantation resulted in a chronic homeostatic expansion of T cells, which acquired effector function after immunosuppression was removed. In contrast, the cytostatic drug mycophenolate mofetil efficiently blocked homeostatic T cell expansion. We propose that the increased production of cytokines that induce homeostatic expansion could contribute to recurrent autoimmunity in transplanted patients with autoimmune disease and that therapy that prevents the expansion of autoreactive T cells will improve the outcome of islet transplantation.
PMCID: PMC2323193  PMID: 18431516
24.  Mature high-affinity immune responses to (pro)insulin anticipate the autoimmune cascade that leads to type 1 diabetes 
Journal of Clinical Investigation  2004;114(4):589-597.
Children at risk for type 1 diabetes can develop early insulin autoantibodies (IAAs). Many, but not all, of these children subsequently develop multiple islet autoantibodies and diabetes. To determine whether disease progression is reflected by autoantibody maturity, IAA affinity was measured by competitive radiobinding assay in first and subsequent IAA-positive samples from children followed from birth in the BABYDIAB cohort. IAA affinity in first positive samples ranged from less than 106 l/mol to more than 1011 l/mol. High affinity was associated with HLA DRB1*04, young age of IAA appearance, and subsequent progression to multiple islet autoantibodies or type 1 diabetes. IAA affinity in multiple antibody–positive children was on average 100-fold higher than in children who remained single IAA positive or became autoantibody negative. All high-affinity IAAs required conservation of human insulin A chain residues 8–13 and were reactive with proinsulin. In contrast, most lower-affinity IAAs were dependent on COOH-terminal B chain residues and did not bind proinsulin. These data are consistent with the concept that type 1 diabetes is associated with sustained early exposure to (pro)insulin in the context of HLA DR4 and show that high-affinity proinsulin-reactive IAAs identify children with the highest diabetes risk.
PMCID: PMC503771  PMID: 15314696

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