Estimation of Individual Positive Anti-Islet Autoantibodies from 3 Screen ICA Titer.

The 3 Screen ICA ELISA is a novel assay capable of simultaneously measuring autoantibodies to glutamic acid decarboxylase (GADA), insulinoma-associated antigen-2 (IA-2A), and zinc transporter 8 (ZnT8A), making it a valuable tool for screening type 1 diabetes. Despite its advantages, it cannot specify which individual autoantibodies are positive or negative. This study aimed to estimate individual positive autoantibodies based on the 3 Screen ICA titer. Six hundred seventeen patients with type 1 diabetes, simultaneously measured for 3 Screen ICA and three individual autoantibodies, were divided into five groups based on their 3 Screen ICA titer. The sensitivities and contribution rates of the individual autoantibodies were then examined. The study had a cross-sectional design. Sixty-nine percent (424 of 617) of patients with type 1 diabetes had 3 Screen ICA titers exceeding the 99th percentile cut-off level (20 index). The prevalence of GADA ranged from 80% to 100% in patients with a 3 Screen ICA over 30 index and 97% of patients with a 3 Screen ICA ≥300 index. Furthermore, the prevalence of all individual autoantibodies being positive was 0% for ≤80 index and as high as 92% for ≥300 index. Significant associations were observed in specific titer groups: the 20-29.9 index group when all the individual autoantibodies were negative, the 30-79.9 index group when positive for GADA alone or IA-2A alone, the 30-299.9 index group when positive for ZnT8A alone, the 80-299.9 index group when positive for both IA-2A and ZnT8A, the 300-499.9 index group when positive for both GADA and ZnT8A, and the ≥300 index group when positive for all individual autoantibodies. These results suggest that the 3 Screen ICA titer may be helpful in estimating individual positive autoantibodies.

SARS-CoV-2 Positive Serology and Islet Autoantibodies in Newly Diagnosed Pediatric Cases of Type 1 Diabetes Mellitus: A Single-Center Cohort Study.

Acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, although presenting less severe forms of the disease in children, seems to play a role in the development of other conditions, including type 1 diabetes mellitus (T1DM). After the beginning of the pandemic, an increase in the number of T1DM pediatric patients was observed in several countries, thus leading to many questions about the complex relationship between SARS-CoV-2 infection and T1DM. Our study aimed to highlight possible correlations between SARS-CoV-2 serology and T1DM onset. Therefore, we performed an observational retrospective cohort study that included 158 children diagnosed with T1DM in the period April 2021-April 2022. The presence or absence of SARS-CoV-2 and T1DM-specific antibodies and other laboratory findings were assessed. In the group of patients with positive SARS-CoV-2 serology, a higher percentage had detectable IA-2A antibodies, more children were positive for all three islet autoantibodies determined (GADA, ICA, and IA-2A), and a higher mean HbA1c value was found. No difference existed between the two groups regarding DKA presence and severity. A lower C-peptide level was found in the patients presenting diabetic ketoacidosis (DKA) at T1DM onset. When compared to a group of patients diagnosed before the pandemic, an increased incidence of both DKA and severe DKA, as well as a higher age at diagnosis and higher levels of HbA1c were present in our study group. These findings have important implications for the ongoing monitoring and management of children with T1DM after the COVID-19 pandemic and highlight the need for further research to better understand the complex relationship between SARS-CoV-2 infection and T1DM.

A multicenter noninferior randomized controlled study comparing the efficacy of laparoscopic versus abdominal radical hysterectomy for cervical cancer (stage IA1 with LVSI, IA2): study protocol of the LAUNCH 1 trial.

BACKGROUND: A retrospective study and a randomized controlled trial published in a high quality journal in late 2018 have shown that laparoscopic radical hysterectomy (RH) was associated with worse survival than abdominal RH among patients with early stage cervical cancer. Radical hysterectomy in cervical cancer has been a classic landmark surgery in gynecology, therefore this conclusion is pivotal. The current trial is designed to reconfirm whether there is a difference between laparoscopic RH and abdominal RH in cervical cancer (stage IA1 with LVSI, IA2) patient survival under stringent operation standards and consistent tumor-free technique. This paper reports the rationale, design, and implementation of the trial. METHODS: This is an investigator-initiated, prospective, randomized, open, blinded endpoint (PROBE) controlled trial. A total of 690 patients with stage IA1 (with intravascular), and IA2 cervical cancer will be enrolled over a period of three years. Patients are randomized (1:1) to either the laparoscopic RH or the abdominal RH group. Patients will then be followed-up for at least five years. The primary endpoint will be 5-year progression-free survival. Secondary endpoints will include 5-year overall survival rates, recurrence rates, operation time, intraoperative blood loss, surgery-related complications, and quality of life. DISCUSSION: The results of the trial will provide valuable evidence for guiding clinical decision of choosing appropriate treatment strategies for stage IA1 (LVSI) and stage IA2 cervical cancer patients. TRIAL REGISTRATION: ClinicalTrials.gov ( NCT04934982 , Registered on 22 June 2021).

HbA1c as a time predictive biomarker for an additional islet autoantibody and type 1 diabetes in seroconverted TEDDY children.

OBJECTIVE: Increased level of glycated hemoglobin (HbA1c) is associated with type 1 diabetes onset that in turn is preceded by one to several autoantibodies against the pancreatic islet beta cell autoantigens; insulin (IA), glutamic acid decarboxylase (GAD), islet antigen-2 (IA-2) and zinc transporter 8 (ZnT8). The risk for type 1 diabetes diagnosis increases by autoantibody number. Biomarkers predicting the development of a second or a subsequent autoantibody and type 1 diabetes are needed to predict disease stages and improve secondary prevention trials. This study aimed to investigate whether HbA1c possibly predicts the progression from first to a subsequent autoantibody or type 1 diabetes in healthy children participating in the Environmental Determinants of Diabetes in the Young (TEDDY) study. RESEARCH DESIGN AND METHODS: A joint model was designed to assess the association of longitudinal HbA1c levels with the development of first (insulin or GAD autoantibodies) to a second, second to third, third to fourth autoantibody or type 1 diabetes in healthy children prospectively followed from birth until 15 years of age. RESULTS: It was found that increased levels of HbA1c were associated with a higher risk of type 1 diabetes (HR 1.82, 95% CI [1.57-2.10], p < 0.001) regardless of first appearing autoantibody, autoantibody number or type. A decrease in HbA1c levels was associated with the development of IA-2A as a second autoantibody following GADA (HR 0.85, 95% CI [0.75, 0.97], p = 0.017) and a fourth autoantibody following GADA, IAA and ZnT8A (HR 0.90, 95% CI [0.82, 0.99], p = 0.036). HbA1c trajectory analyses showed a significant increase of HbA1c over time (p < 0.001) and that the increase is more rapid as the number of autoantibodies increased from one to three (p < 0.001). CONCLUSION: In conclusion, increased HbA1c is a reliable time predictive marker for type 1 diabetes onset. The increased rate of increase of HbA1c from first to third autoantibody and the decrease in HbA1c predicting the development of IA-2A are novel findings proving the link between HbA1c and the appearance of autoantibodies.

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 ß-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 ß-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.

Identification of autoimmune type 1 diabetes and multiple organ-specific autoantibodies in adult-onset non-insulin-requiring diabetes in China: A population-based multicentre nationwide survey.

AIMS: To investigate the prevalence of adult-onset autoimmune diabetes (ADM) and predisposition to autoimmune diseases by quantifying serum organ-specific autoantibodies in people with phenotype of type 2 diabetes (T2D). MATERIALS AND METHODS: We included a nationally representative sample of 46 239 adults aged ≥20 years from 14 provinces, of whom 4671 had diabetes, plus 1000 control subjects with normal glucose tolerance (NGT). Participants were screened centrally for autoantibodies to glutamic acid decarboxylase (GAD), islet antigen 2 (IA2) and zinc transporter isoform-8 (Znt8) and were defined as having ADM where positive for these antibodies. We then assayed thyroid peroxidase (TPO), tissue transglutaminase (tTG) and 21-hydroxylase (21-OH) autoantibodies in randomly selected participants with ADM and in age-matched, sex-matched and non-ADM controls with T2D plus controls with NGT. RESULTS: Post-normalization, the standardized prevalence rate of ADM was 6.0% (95% confidence interval [CI] 5.3-6.8) in initially non-insulin-requiring participants with ADM, corresponding to six million adults in China, in whom adjusted antibody positivity was: TPO autoantibodies 16.3% (95% CI 10.8-21.8), tTG autoantibodies 2.1% (95% CI 0.0-4.2), and 21-OH autoantibodies 1.8% (95% CI -0.2 to 3.8). Those participants with ADM who were GAD autoantibody-positive had high risk of TPO autoantibody positivity (odds ratio [OR] 2.39, P = 0.0031) and tTG autoantibody positivity (OR 6.98, P = 0.027), while those positive for IA2 autoantibodies had a high risk of tTG autoantibody positivity (OR 19.05, P = 0.001). CONCLUSIONS: A proportion of people with phenotype of T2D in China have ADM, with diabetes-associated autoantibodies, and may be at risk of developing other organ-specific autoimmune diseases; therefore, it may be clinically relevant to consider screening such Chinese populations.

Gestational respiratory infections interacting with offspring HLA and CTLA-4 modifies incident ß-cell autoantibodies.

ß-cell autoantibodies against insulin (IAA), GAD65 (GADA) and IA-2 (IA-2A) precede onset of childhood type 1 diabetes (T1D). Incidence of the first appearing ß-cell autoantibodies peaks at a young age and is patterned by T1D-associated genes, suggesting an early environmental influence. Here, we tested if gestational infections and interactions with child's human leukocyte antigen (HLA) and non-HLA genes affected the appearance of the first ß-cell autoantibody. Singletons of mothers without diabetes (n = 7472) with T1D-associated HLA-DR-DQ genotypes were prospectively followed quarterly through the first 4 years of life, then semiannually until age 6 years, using standardized autoantibody analyses. Maternal infections during pregnancy were assessed via questionnaire 3-4.5 months post-delivery. Polymorphisms in twelve non-HLA genes associated with the first appearing ß-cell autoantibodies were included in a Cox regression analysis. IAA predominated as the first appearing ß-cell autoantibody in younger children (n = 226, median age at seroconversion 1.8 years) and GADA (n = 212; 3.2 years) in children aged ≥2 years. Gestational infections were not associated with the first appearing ß-cell autoantibodies overall. However, gestational respiratory infections (G-RI) showed a consistent protective influence on IAA (HR 0.64, 95% CI 0.45-0.91) among CTLA4-(AG, GG) children (G-RI*CTLA4 interaction, p = 0.002). The predominant associations of HLA-DR-DQ 4-8/8-4 with IAA and HLA-DR-DQ 3-2/3-2 with GADA were not observed if a G-RI was reported (G-RI*HLA-DR-DQ interaction, p = 0.03). The role of G-RI may depend on offspring HLA and CTLA-4 alleles and supports a bidirectional trigger for IAA or GADA as a first appearing ß-cell autoantibody in early life.

Presence of diabetes-specific autoimmunity in women with gestational diabetes mellitus (GDM) predicts impaired glucose regulation at follow-up.

PURPOSE: Gestational diabetes mellitus (GDM) is the most frequent complication of pregnancy; around 10% of GDM cases may be determined by autoimmunity, and our aims were to establish the role of autoimmunity in a population of Sardinian women affected by GDM, to find predictive factors for autoimmune GDM, and to determine type 1 diabetes (T1D) auto-antibodies (Aabs) together with glucose tolerance after a mean 21.2 months of follow-up. METHODS: We consecutively recruited 143 women affected by GDM and 60 without GDM; clinical data and pregnancy outcomes were obtained by outpatient visit or phone recall. T1D auto-antibodies GADA, IA2-A, IAA, ZnT8-A were measured in the whole population at baseline, and in the Aab-positive women at follow-up. RESULTS: The overall prevalence of autoimmunity was 6.4% (13/203). No significant difference was found in the prevalence of auto-antibodies between GDM (5.6%) and control (8.3%) women, neither in antibody titres. Highest titres for GADA and ZnT8-A were observed in the control group; no phenotypic factors were predictive for autoimmune GDM. Diabetes-related autoantibodies were still present in all the GDM women at follow-up, and their presence was associated with a 2.65 (p < 0.0016) relative risk (RR) of glucose impairment. CONCLUSION: We observed a low prevalence (5.6%) of diabetes-related autoimmunity in our GDM cohort, consistent with the prevalence reported in previous studies. It was not possible to uncover features predictive of autoimmune GDM. However, given the significant risk of a persistent impaired glycemic regulation at follow-up, it is advisable to control for glucose tolerance in GDM women with diabetes-related autoimmunity.

Characterisation of rapid progressors to type 1 diabetes among children with HLA-conferred disease susceptibility.

AIMS/HYPOTHESIS: In this study, we aimed to characterise rapid progressors to type 1 diabetes among children recruited from the general population, on the basis of HLA-conferred disease susceptibility. METHODS: We monitored 7410 HLA-predisposed children participating in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study for the development of beta cell autoimmunity and type 1 diabetes from birth over a median follow-up time of 16.2 years (range 0.9-21.1 years). Islet cell antibodies (ICA) and autoantibodies to insulin (IAA), GAD (GADA) and islet antigen 2 (IA-2A) were assessed as markers of beta cell autoimmunity. Rapid progression was defined as progression to clinical type 1 diabetes within 1.5 years of autoantibody seroconversion. We analysed the association between rapid progression and demographic and autoantibody characteristics as well as genetic markers, including 25 non-HLA SNPs predisposing to type 1 diabetes. RESULTS: Altogether, 1550 children (21%) tested positive for at least one diabetes-associated autoantibody in at least two samples, and 248 (16%) of seroconverters progressed to type 1 diabetes by the end of 2015. The median time from seroconversion to diagnosis was 0.51 years in rapid progressors (n = 42, 17%) and 5.4 years in slower progressors. Rapid progression was observed both among young (<5 years) and early pubertal children (>7 years), resulting in a double-peak distribution of seroconversion age. Compared with slower progressors, rapid progressors had a higher frequency of positivity for multiple (≥2) autoantibodies and had higher titres of ICA, IAA and IA-2A at seroconversion, and there was a higher prevalence of the secretor genotype in the FUT2 gene among those carrying the high-risk HLA genotype. Compared with autoantibody-positive non-progressors, rapid progressors were younger, were more likely to carry the high-risk HLA genotype and a predisposing SNP in the PTPN22 gene, had higher frequency of ICA, IAA, GADA and IA-2A positivity and multipositivity, and had higher titres of all four autoantibodies at seroconversion. CONCLUSIONS/INTERPRETATION: At seroconversion, individuals with rapid progression to type 1 diabetes were characterised by a younger age, higher autoantibody titres, positivity for multiple autoantibodies and higher prevalence of a FUT2 SNP. The double-peak profile for seroconversion age among the rapid progressors demonstrates for the first time that rapid progression may take place not only in young children but also in children in early puberty. Rapid progressors might benefit from careful clinical follow-up and early preventive measures.

Biochemical, biophysical, and functional properties of ICA512/IA-2 RESP18 homology domain.

BACKGROUND: ICA512 (or IA-2/PTPRN) is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Previous studies implied its involvement in generation, cargo storage, traffic, exocytosis and recycling of insulin secretory granules, as well as in ß-cell proliferation. While several ICA512 domains have been characterized, the function and structure of a large portion of its N-terminal extracellular (or lumenal) region are unknown. Here, we report a biophysical, biochemical, and functional characterization of ICA512-RESP18HD, a domain comprising residues 35 to 131 and homologous to regulated endocrine-specific protein 18 (RESP18). METHODS: Pure recombinant ICA512-RESP18HD was characterized by CD and fluorescence. Its binding to insulin and proinsulin was characterized by ELISA, surface plasmon resonance, and fluorescence anisotropy. Thiol reactivity was measured kinetically. Targeting of ΔRESP18HD ICA512-GFP to the membrane of insulinoma cells was monitored by immunofluorescence. RESULTS: ICA512-RESP18HD possesses a strong tendency to aggregate and polymerize via intermolecular disulfide formation, particularly at pH>4.5. Its cysteine residues are highly susceptible to oxidation forming an intramolecular disulfide between cysteine 53 and 62 and intermolecular disulfides via cysteine 40 and cysteine 47. The regulated sorting of ICA512 to secretory granules in INS-1 cells was impaired by deletion of RESP18HD. ICA512-RESP18HD binds with high-affinity to insulin and proinsulin. CONCLUSIONS: RESP18HD is required for efficient sorting of ICA512 to secretory granules. GENERAL SIGNIFICANCE: RESP18HD is a key determinant for ICA512 granule targeting.

Influence of HLA-DR and -DQ alleles on autoantibody recognition of distinct epitopes within the juxtamembrane domain of the IA-2 autoantigen in type 1 diabetes.

AIMS/HYPOTHESIS: Insulinoma-associated protein 2 (IA-2) is a major target of autoimmunity in type 1 diabetes. When first detected, IA-2-autoantibodies commonly bind epitopes in the juxtamembrane (JM) domain of IA-2 and antibody responses subsequently spread to the tyrosine phosphatase domain. Definition of structures of epitopes in the JM domain, and genetic requirements for autoimmunity to these epitopes, is important for our understanding of initiation and progression of autoimmunity. The aims of this study were to investigate the contribution of individual amino acids in the IA-2 JM domain to antibody binding to these epitopes and the role of HLA genotypes in determining epitope specificity. METHODS: Regions of the JM domain recognised by autoantibodies were identified by peptide competition and inhibitory effects of alanine substitutions of residues within the JM region. Antibody binding was determined by radioligand binding assays using sera from patients genotyped for HLA-DRB1 and -DQB1 alleles. RESULTS: Patients were categorised into two distinct groups of JM antibody reactivity according to peptide inhibition. Inhibition by substitutions of individual amino acids within the JM domain differed between patients, indicating heterogeneity in epitope recognition. Cluster analysis defined six groups of residues having similar inhibitory effects on antibody binding, with three clusters showing differences in patients affected or unaffected by peptide. One cluster demonstrated significant differences in antibody binding between HLA-DRB1*04 and HLA-DRB1*07 patients and within DRB1*04 individuals; antibody recognition of a second cluster depended on expression of HLA-DQB1*0302. CONCLUSIONS/INTERPRETATION: The results identify amino acids contributing to distinct epitopes on IA-2, with both HLA-DR and HLA-DQ alleles influencing epitope specificity.

The implications of autoantibodies to a single islet antigen in relatives with normal glucose tolerance: development of other autoantibodies and progression to type 1 diabetes.

AIMS/HYPOTHESIS: Autoantibodies directed at single islet autoantigens are associated with lower overall risk of type 1 diabetes than multiple autoantibodies, but individuals with one autoantibody may progress to higher risk categories. We examined the characteristics of this progression in relatives followed prospectively in the TrialNet Pathway to Prevention. METHODS: The study population comprised 983 relatives who were single autoantibody positive with normal baseline glucose tolerance (median age 16.2 years). Samples were screened for antibodies to GAD, insulinoma-associated antigen 2 (IA-2) and insulin, and all positive samples tested for antibodies to zinc transporter 8 and islet cell antibodies. RESULTS: Antibodies to at least one additional islet autoantigen appeared in 118 of 983 relatives (overall 5 year risk 22%, 95% CI [17.9, 26.1]). At baseline, antibodies to GAD alone (68%) were more frequent than antibodies to insulin (26%) or IA-2 (6%), but all were associated with a similar risk of developing additional autoantibodies. Risk was associated with younger age (p = 0.002) and HLA class II genotype, but was similar in high and intermediate genetic risk groups (p = 0.65). Relatives who became multiple autoantibody positive during the follow-up had increased risk of developing diabetes comparable with the risk in relatives with multiple autoantibodies at study entry. CONCLUSIONS/INTERPRETATION: Progression of islet autoimmunity in single autoantibody positive relatives in late childhood/adult life is associated with a predominance of autoantibodies to GAD and a distinct HLA risk profile. This heterogeneity in type 1 diabetes autoimmunity has potentially important implications for disease prevention.

Novel prokaryotic expression of thioredoxin-fused insulinoma associated protein tyrosine phosphatase 2 (IA-2), its characterization and immunodiagnostic application.

BACKGROUND: The insulinoma associated protein tyrosine phosphatase 2 (IA-2) is one of the immunodominant autoantigens involved in the autoimmune attack to the beta-cell in Type 1 Diabetes Mellitus. In this work we have developed a complete and original process for the production and recovery of the properly folded intracellular domain of IA-2 fused to thioredoxin (TrxIA-2ic) in Escherichia coli GI698 and GI724 strains. We have also carried out the biochemical and immunochemical characterization of TrxIA-2icand design variants of non-radiometric immunoassays for the efficient detection of IA-2 autoantibodies (IA-2A). RESULTS: The main findings can be summarized in the following statements: i) TrxIA-2ic expression after 3 h of induction on GI724 strain yielded ≈ 10 mg of highly pure TrxIA-2ic/L of culture medium by a single step purification by affinity chromatography, ii) the molecular weight of TrxIA-2ic (55,358 Da) could be estimated by SDS-PAGE, size exclusion chromatography and mass spectrometry, iii) TrxIA-2ic was properly identified by western blot and mass spectrometric analysis of proteolytic digestions (63.25 % total coverage), iv) excellent immunochemical behavior of properly folded full TrxIA-2ic was legitimized by inhibition or displacement of [35S]IA-2 binding from IA-2A present in Argentinian Type 1 Diabetic patients, v) great stability over time was found under proper storage conditions and vi) low cost and environmentally harmless ELISA methods for IA-2A assessment were developed, with colorimetric or chemiluminescent detection. CONCLUSIONS: E. coli GI724 strain emerged as a handy source of recombinant IA-2ic, achieving high levels of expression as a thioredoxin fusion protein, adequately validated and applicable to the development of innovative and cost-effective immunoassays for IA-2A detection in most laboratories.

Islet Autoantibodies.

Islet autoantibodies are the main markers of pancreatic autoimmunity in type 1 diabetes (T1D). Islet autoantibodies recognize insulin (IAA), glutamic acid decarboxylase (GADA), protein phosphatase-like IA-2 (IA-2A), and ZnT8 (ZnT8A), all antigens that are found on secretory granules within pancreatic beta cells. Islet antibodies, measured by sensitive and specific liquid phase assays, are the key parameters of the autoimmune response monitored for diagnostics or prognostics in patients with T1D or for disease prediction in at-risk individuals before T1D onset. Islet autoantibodies have been the main tool used to explore the natural history of T1D; this review summarizes the current knowledge about the autoantigens and the phenotype of islets autoantibodies acquired in large prospective studies from birth in children at risk of developing T1D.

Environmental factors in the etiology of type 1 diabetes, celiac disease, and narcolepsy.

The etiology of human leukocyte antigen (HLA)-associated organ-specific autoimmune diseases is incomplete. In type 1 diabetes and celiac disease, the strongest associations are with the HLA-DR3-DQ2 and DR4-DQ8 haplotypes, whereas the DQB1*06:02 allele has a strong negative association. In contrast, narcolepsy, especially as recently triggered by the Pandemrix(®) H1N1 vaccine (GlaxoKlineSmith (GSK), Brentford, Middlesex, UK), did not seem to develop without at least one copy of the latter allele. The overall hypothesis is that the role of these different HLA haplotypes, especially in Finland and Sweden, is related to the immune response to infectious agents that are common in these two populations. The high incidence of both type 1 diabetes and celiac disease in Scandinavia may be the result of the HLA-DR3-DQ2 and DR4-DQ8 haplotypes, and the DQB1*06:02 allele are common because they protected people from succumbing to common infections. The timing of dissecting the autoimmune response is critical to understand the possible role of environmental factors. First, an etiological trigger may be a common virus infecting beta cells or with antigens inducing beta-cell cross reactivity. Second, an autoimmune reaction may ensue, perhaps in response to beta-cell apoptosis or autophagy, resulting in autoantigen-specific T cells and autoantibodies. It is critical in at-risk children to dissect the immune response prior to the appearance of autoantibodies in order to identify cellular reactions in response to environmental factors that are able to induce an HLA-associated immune reaction.

The Ia-2ß intronic miRNA, miR-153, is a negative regulator of insulin and dopamine secretion through its effect on the Cacna1c gene in mice.

AIMS/HYPOTHESIS: miR-153 is an intronic miRNA embedded in the genes that encode IA-2 (also known as PTPRN) and IA-2ß (also known as PTPRN2). Islet antigen (IA)-2 and IA-2ß are major autoantigens in type 1 diabetes and are important transmembrane proteins in dense core and synaptic vesicles. miR-153 and its host genes are co-regulated in pancreas and brain. The present experiments were initiated to decipher the regulatory network between miR-153 and its host gene Ia-2ß (also known as Ptprn2). METHODS: Insulin secretion was determined by ELISA. Identification of miRNA targets was assessed using luciferase assays and by quantitative real-time PCR and western blots in vitro and in vivo. Target protector was also employed to evaluate miRNA target function. RESULTS: Functional studies revealed that miR-153 mimic suppresses both glucose- and potassium-induced insulin secretion (GSIS and PSIS, respectively), whereas miR-153 inhibitor enhances both GSIS and PSIS. A similar effect on dopamine secretion also was observed. Using miRNA target prediction software, we found that miR-153 is predicted to target the 3'UTR region of the calcium channel gene, Cacna1c. Further studies confirmed that Cacna1c mRNA and protein are downregulated by miR-153 mimics and upregulated by miR-153 inhibitors in insulin-secreting freshly isolated mouse islets, in the insulin-secreting mouse cell line MIN6 and in the dopamine-secreting cell line PC12. CONCLUSIONS/INTERPRETATION: miR-153 is a negative regulator of both insulin and dopamine secretion through its effect on Cacna1c expression, which suggests that IA-2ß and miR-153 have opposite functional effects on the secretory pathway.

The cytokine production of peripheral blood mononuclear cells reflects the autoantibody profile of patients suffering from type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disorder characterised by the immune-mediated destruction of insulin-producing pancreatic beta cells. The inflammatory process appears to be primarily mediated by pro-inflammatory Th1 lymphocytes, while the role Th17 cells in T1D is currently being investigated. T1D is characterised by the presence of autoantigen-specific autoantibodies. This study was conducted using patients with confirmed T1D and healthy control subjects. We examined the effect of the patient's autoantibody profile on peripheral blood mononuclear cell (PBMC) cytokine production following stimulation with the major diabetogenic autoantigens GAD65 and IA2. IFN-gamma and IL17 production was detected by ELISPOT and the ratio of basic cellular populations in PBMCs was measured by flow cytometry. We demonstrated a significant interaction between the patient's autoantibody profile and mode of stimulation. This suggests that autoantigen stimulation has a different effect on different groups of patients depending on their autoantibody profile. An increased production of IL17 was found in patients with high IA2 autoantibodies compared to patients with low levels of autoantibodies and healthy controls regardless of the mode of stimulation. The titre of IA2 autoantibodies positively correlates with the proportion of Tc lymphocytes and negatively correlates with the proportion of Th lymphocytes. Our results show that a patient's autoantibody profile reflects the type of cellular immune responses. It seems that the high titre of IA2 autoantibodies is related to increased production of IL17 and an increased proportion of Tc lymphocytes. This finding may be useful in designing immunointervention studies to prevent T1D.

Analysis of the autoimmune regulator gene in patients with autoimmune non-APECED polyendocrinopathies.

The pathogenesis of autoimmunity was derived from a complex interaction of genetic and environmental factors. Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy is a rare autosomal recessive disease caused by mutations in the autoimmune regulator (AIRE) gene. AIRE gene variants and, in particular, heterozygous loss-of-function mutations were also discovered in organ-specific autoimmune disorders, possibly contributing to their etiopathogenesis. It was suggested that even predisposition to develop certain autoimmune conditions may be derived from AIRE gene polymorphisms including S278R and intronic IVS9+6 G>A. In this study we unravel the hypothesis on whether AIRE gene variants may predispose individuals to associated autoimmune conditions in 41 Italian patients affected by non-APECED autoimmune polyendocrinopathies. We could not detect any heterozygous mutations of the AIRE gene. Although a trend of association was observed, heterozygous polymorphisms S278R and IVS9+6 G>A were detected in patients without statistically significant prevalence than in controls. Their putative contribution to autoimmune polyendocrinopathies and their predictive value in clinical strategies of disease development could be unravelled by analysing a larger sample of diseased patients and healthy individuals.

Childhood screening for type 1 diabetes comparing automated multiplex Antibody Detection by Agglutination-PCR (ADAP) with single plex islet autoantibody radiobinding assays.

BACKGROUND: Two or more autoantibodies against either insulin (IAA), glutamic acid decarboxylase (GADA), islet antigen-2 (IA-2A) or zinc transporter 8 (ZnT8A) denote stage 1 (normoglycemia) or stage 2 (dysglycemia) type 1 diabetes prior to stage 3 type 1 diabetes. Automated multiplex Antibody Detection by Agglutination-PCR (ADAP) assays in two laboratories were compared to single plex radiobinding assays (RBA) to define threshold levels for diagnostic specificity and sensitivity. METHODS: IAA, GADA, IA-2A and ZnT8A were analysed in 1504 (54% females) population based controls (PBC), 456 (55% females) doctor's office controls (DOC) and 535 (41% females) blood donor controls (BDC) as well as in 2300 (48% females) patients newly diagnosed (1-10 years of age) with stage 3 type 1 diabetes. The thresholds for autoantibody positivity were computed in 100 10-fold cross-validations to separate patients from controls either by maximizing the χ2-statistics (chisq) or using the 98th percentile of specificity (Spec98). Mean and 95% CI for threshold, sensitivity and specificity are presented. FINDINGS: The ADAP ROC curves of the four autoantibodies showed comparable AUC in the two ADAP laboratories and were higher than RBA. Detection of two or more autoantibodies using chisq showed 0.97 (0.95, 0.99) sensitivity and 0.94 (0.91, 0.97) specificity in ADAP compared to 0.90 (0.88, 0.95) sensitivity and 0.97 (0.94, 0.98) specificity in RBA. Using Spec98, ADAP showed 0.92 (0.89, 0.95) sensitivity and 0.99 (0.98, 1.00) specificity compared to 0.89 (0.77, 0.86) sensitivity and 1.00 (0.99, 1.00) specificity in the RBA. The diagnostic sensitivity and specificity were higher in PBC compared to DOC and BDC. INTERPRETATION: ADAP was comparable in two laboratories, both comparable to or better than RBA, to define threshold levels for two or more autoantibodies to stage type 1 diabetes. FUNDING: Supported by The Leona M. and Harry B. Helmsley Charitable Trust (grant number 2009-04078), the Swedish Foundation for Strategic Research (Dnr IRC15-0067) and the Swedish Research Council, Strategic Research Area (Dnr 2009-1039). AL was supported by the DiaUnion collaborative study, co-financed by EU Interreg ÖKS, Capital Region of Denmark, Region Skåne and the Novo Nordisk Foundation.

Reaching the Diagnosis of Checkpoint Inhibitor-Induced Diabetes Mellitus in Different Clinical Scenarios: A Real-World Application of Updated Diagnostic Criteria.

BACKGROUND: Checkpoint inhibitor (CPI)-associated diabetes mellitus (CPI-DM) is a rare immune-related adverse event (irAE) that presents with variable clinical manifestations. Data about its pathogenesis have not yet been adequately studied. METHODS: Applying the recently updated diagnostic criteria from the American Diabetes Association, we retrospectively reviewed the medical records of all CPI-treated patients referred to our endocrinological unit for managing their endocrine irAEs and analyzed the incidence of CPI-DM, its clinical characteristics, and its management. RESULTS: Among the 326 CPI-treated patients with endocrine irAEs, 4 patients met the updated criteria for the diagnosis of CPI-DM, representing 1.22% of all endocrine irAEs in our cohort. These four patients presented with distinct clinical scenarios regarding the irAE onset, the underlying malignancy, the administered CPI regimen, and the type of circulating autoantibodies. CONCLUSION: The variable presentation of CPI-DM and the non-standard sensitivity of the presence of the type 1 DM traditional autoantibodies highlight the need for distinct guidelines and increased awareness of its diagnosis and management.