RESUMEN
AIMS: In recent-onset type 1 diabetes, clamp-derived C-peptide predicts good response to anti-CD3. Elevated proinsulin and proinsulin/C-peptide ratio (PI/CP) suggest increased metabolic/inflammatory beta cell burden. We reanalyzed trial data to compare the ability of baseline acutely glucose-stimulated proinsulin, C-peptide and PI/CP to predict functional outcome. METHODS: Eighty recent-onset type 1 diabetes patients participated in the placebo-controlled otelixizumab (GSK; NCT00627146) trial. Hyperglycemic clamps were performed at baseline, 6, 12 and 18 months, involving 3 h of induced euglycemia, followed by acutely raising and maintaining glycemia to ≥ 10 mmol/l for 140 min. Plasma proinsulin, C-peptide and PI/CP were determined after acute (minute 0 at 10 mmol/l; PI0, CP0, PI/CP0) and sustained glucose stimulation (AUC between minutes 60-140). Outcome was assessed as change in AUC60-140 C-peptide from baseline. RESULTS: In multiple linear regression, higher baseline (≥median [P50]) PI0 independently predicted preservation of beta cell function in response to anti-CD3 and interacted significantly with IAA. During follow-up, anti-CD3 tempered a further increase in PI/CP0, but not in PI0. CP0 outperformed PI0 and PI/CP0 for post-treatment monitoring. CONCLUSIONS: In recent-onset type 1 diabetes, elevated acutely glucose-stimulated proinsulin may complement or replace acutely or sustainedly stimulated C-peptide release for identifying good responders to anti-CD3, but not as outcome measure.
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Diabetes Mellitus Tipo 1 , Proinsulina , Humanos , Proinsulina/metabolismo , Proinsulina/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Insulina/uso terapéutico , Glucosa/uso terapéutico , Péptido C , Glucemia/metabolismoRESUMEN
The HLA region is the major genetic risk determinant of Type 1 diabetes. How non-HLA loci contribute to the genetic risk is incompletely understood, but there are indications that at least some impact progression of asymptomatic autoimmunity. We examined whether SNPs in 7 susceptibility loci (INS, SH2B3, PTPN2, PTPN22, CTLA4, CLEC16A, and IL2RA) could improve prediction of the progression from single to multiple autoantibody positivity, and from there on to diagnosis. SNPs were genotyped in persistently autoantibody positive relatives by allelic discrimination qPCR and disease progression was studied by multivariate Cox regression analysis. In our cohort, only the CTLA4 GA genotype (rs3087243, P = 0.002) and the CLEC16A AA genotype (rs12708716, P = 0.021) were associated with accelerated progression from single to multiple autoantibody positivity, but their effects were restricted to presence of HLA-DQ2/DQ8, and IAA as first autoantibody, respectively. The interaction of CTLA4 and HLA-DQ2/DQ8 overruled the effect of DQ2/DQ8 alone. The HLA-DQ2/DQ8-mediated risk of progression to multiple autoantibodies nearly entirely depended on heterozygosity for CTLA4. The SH2B3 TT genotype (rs3184504) was protective for HLA-DQ8 positive subjects (P = 0.003). At the stage of multiple autoantibodies, only the CTLA4 GA genotype was a minor independent risk factor for progression towards clinical diabetes (P = 0.034). Our study shows that non-HLA polymorphisms impact progression of islet autoimmunity in a subgroup-, stage- and SNP-specific way, suggesting distinct mechanisms. If confirmed, these findings may help refine risk assessment, follow-up, and prevention trials in risk groups.
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Diabetes Mellitus Tipo 1 , Humanos , Autoanticuerpos , Autoinmunidad/genética , Antígeno CTLA-4/genética , Diabetes Mellitus Tipo 1/genética , Predisposición Genética a la Enfermedad , Genotipo , Lectinas Tipo C/genética , Proteínas de Transporte de Monosacáridos/genética , Polimorfismo de Nucleótido Simple , Proteína Tirosina Fosfatasa no Receptora Tipo 22/genéticaRESUMEN
AIMS/HYPOTHESIS: We examined whether the non-HLA susceptibility locus ERBB3/IKZF4 influences progression of type 1 diabetes stage specifically according to sex. METHODS: SNPs of ERBB3 (rs2292239 T/G) and IKZF4 (rs1701704 G/T) were screened by allelic discrimination quantitative PCR assay in first-degree relatives of type 1 diabetes patients who had developed at least one circulating autoantibody. The effect of ERBB3/IKZF4 genotypes and sex, on the progression of single autoantibody positivity to multiple autoantibody positivity and from multiple autoantibody positivity to diabetes, was studied by Kaplan-Meier analysis and multivariate Cox regression. RESULTS: In the cohort of autoantibody-positive first-degree relatives, the risk allele frequencies for ERBB3 rs2292239 (T) and IKZF4 rs1701704 (G) were increased. There was a significant male excess at the stage of multiple autoantibody positivity (p = 0.021). In Kaplan-Meier survival analysis, progression from single to multiple antibody positivity was delayed in female participants with genotype ERBB3 GG (p = 0.018, vs ERBB3 TG+TT) or IKZF4 TT (p = 0.023, vs IKZF4 GT+GG), but not in male participants. In multivariate Cox regression models, the interaction effects between female sex and ERBB3 GG (p = 0.012; HR = 0.305 [95% CI 0.120, 0.773]) or between female sex and IKZF4 TT (p = 0.011; HR = 0.329 [95% CI 0.140, 0.777]) emerged as potential determinants of delayed progression to multiple autoantibodies. The progression from multiple autoantibody positivity to type 1 diabetes appeared not to be influenced by ERBB3/IKZF4. CONCLUSIONS/INTERPRETATION: In siblings and offspring of type 1 diabetes patients, polymorphism in region ERBB3/IKZF4 may affect disease progression at the level of epitope spreading in female individuals. Our findings suggest that interaction between sex and ERBB3/IKZF4 may contribute to the post-pubertal male excess in type 1 diabetes.
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Autoanticuerpos/sangre , Autoantígenos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Epítopos/inmunología , Factor de Transcripción Ikaros/genética , Receptor ErbB-3/genética , Caracteres Sexuales , Adolescente , Adulto , Niño , Diabetes Mellitus Tipo 1/genética , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Humanos , Insulina/inmunología , Masculino , Polimorfismo de Nucleótido Simple/genética , Modelos de Riesgos Proporcionales , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Tirosina Fosfatasas Clase 8 Similares a Receptores/inmunología , Transportador 8 de Zinc/inmunologíaRESUMEN
Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.
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Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/patología , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Trasplante de Islotes Pancreáticos , MicroARNs/genética , Biomarcadores/metabolismo , Recuento de Células , Estudios de Cohortes , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , MicroARNs/metabolismo , Curva ROC , Reproducibilidad de los Resultados , TropismoRESUMEN
Autoantibodies against islet cell antigens are routinely used to identify subjects at increased risk of symptomatic type 1 diabetes, but their relation to the intra-islet pathogenetic process that leads to positivity for these markers is poorly understood. We screened 556 non-diabetic organ donors (3 months to 24 years) for five different autoantibodies and found positivity in 27 subjects, 25 single- and two double autoantibody-positive donors. Histopathological screening of pancreatic tissue samples showed lesion characteristic for recent-onset type 1 diabetes in the two organ donors with a high-risk profile, due to their positivity for multiple autoantibodies and HLA-inferred risk. Inflammatory infiltrates (insulitis) were found in a small fraction of islets (<5%) and consisted predominantly of CD3+CD8+ T-cells. Islets with insulitis were found in close proximity to islets devoid of insulin-positivity; such pseudo-atrophic islets were present in multiple small foci scattered throughout the pancreatic tissue or were found to be distributed with a lobular pattern. Relative beta cell area in both single and multiple autoantibody-positive donors was comparable to that in autoantibody-negative controls. In conclusion, in organ donors under age 25 years, insulitis and pseudo-atrophic islets were restricted to multiple autoantibody-positive individuals allegedly at high risk of developing symptomatic type 1 diabetes, in line with reports in older age groups. These observations may give further insight into the early pathogenetic events that may culminate in clinically overt disease.
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Antígenos/inmunología , Autoanticuerpos/sangre , Linfocitos T CD8-positivos/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Trasplante de Islotes Pancreáticos , Donantes de Tejidos , Adolescente , Factores de Edad , Biomarcadores/sangre , Estudios de Casos y Controles , Proliferación Celular , Niño , Preescolar , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/patología , Selección de Donante , Femenino , Humanos , Lactante , Células Secretoras de Insulina/patología , Masculino , Adulto JovenRESUMEN
AIMS/HYPOTHESIS: Numerous clinical studies have investigated the anti-CD3É monoclonal antibody otelixizumab in individuals with type 1 diabetes, but limited progress has been made in identifying the optimal clinical dose with acceptable tolerability and safety. The aim of this study was to evaluate the association between dose-response, safety and tolerability, beta cell function preservation and the immunological effects of otelixizumab in new-onset type 1 diabetes. METHODS: In this randomised, single-blind, placebo-controlled, 24 month study, conducted in five centres in Belgium via the Belgian Diabetes Registry, participants (16-27 years old, <32 days from diagnosis of type 1 diabetes) were scheduled to receive placebo or otelixizumab in one of four dose cohorts (cumulative i.v. dose 9, 18, 27 or 36 mg over 6 days; planned n = 40). Randomisation to treatment was by a central computer system; only participants and bedside study personnel were blinded to study treatment. The co-primary endpoints were the incidence of adverse events, the rate of Epstein-Barr virus (EBV) reactivation, and laboratory measures and vital signs. A mixed-meal tolerance test was used to assess beta cell function; exploratory biomarkers were used to measure T cell responses. RESULTS: Thirty participants were randomised/28 were analysed (placebo, n = 6/5; otelixizumab 9 mg, n = 9/8; otelixizumab 18 mg, n = 8/8; otelixizumab 27 mg, n = 7/7; otelixizumab 36 mg, n = 0). Dosing was stopped at otelixizumab 27 mg as the predefined EBV reactivation stopping criteria were met. Adverse event frequency and severity were dose dependent; all participants on otelixizumab experienced at least one adverse event related to cytokine release syndrome during the dosing period. EBV reactivation (otelixizumab 9 mg, n = 2/9; 18 mg, n = 4/8: 27 mg, n = 5/7) and clinical manifestations (otelixizumab 9 mg, n = 0/9; 18 mg, n = 1/8; 27 mg, n = 3/7) were rapid, dose dependent and transient, and were associated with increased productive T cell clonality that diminished over time. Change from baseline mixed-meal tolerance test C-peptide weighted mean AUC0-120 min following otelixizumab 9 mg was above baseline for up to 18 months (difference from placebo 0.39 [95% CI 0.06, 0.72]; p = 0.023); no beta cell function preservation was observed at otelixizumab 18 and 27 mg. CONCLUSIONS/INTERPRETATION: A metabolic response was observed with otelixizumab 9 mg, while doses higher than 18 mg increased the risk of unwanted clinical EBV reactivation. Although otelixizumab can temporarily compromise immunocompetence, allowing EBV to reactivate, the effect is dose dependent and transient, as evidenced by a rapid emergence of EBV-specific T cells preceding long-term control over EBV reactivation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02000817. FUNDING: The study was funded by GlaxoSmithKline. Graphical abstract.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Adolescente , Adulto , Péptido C/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Progresión de la Enfermedad , Relación Dosis-Respuesta a Droga , Infecciones por Virus de Epstein-Barr/inducido químicamente , Femenino , Humanos , Infección Latente/inducido químicamente , Masculino , Método Simple Ciego , Adulto JovenRESUMEN
At diagnosis, most people with type 1 diabetes (T1D) produce measurable levels of endogenous insulin, but the rate at which insulin secretion declines is heterogeneous. To explain this heterogeneity, we sought to identify a composite signature predictive of insulin secretion, using a collaborative assay evaluation and analysis pipeline that incorporated multiple cellular and serum measures reflecting ß cell health and immune system activity. The ability to predict decline in insulin secretion would be useful for patient stratification for clinical trial enrollment or therapeutic selection. Analytes from 12 qualified assays were measured in shared samples from subjects newly diagnosed with T1D. We developed a computational tool (DIFAcTO, Data Integration Flexible to Account for different Types of data and Outcomes) to identify a composite panel associated with decline in insulin secretion over 2 years following diagnosis. DIFAcTO uses multiple filtering steps to reduce data dimensionality, incorporates error estimation techniques including cross-validation and sensitivity analysis, and is flexible to assay type, clinical outcome, and disease setting. Using this novel analytical tool, we identified a panel of immune markers that, in combination, are highly associated with loss of insulin secretion. The methods used here represent a potentially novel process for identifying combined immune signatures that predict outcomes relevant for complex and heterogeneous diseases like T1D.
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Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/inmunología , Progresión de la Enfermedad , Secreción de Insulina/fisiología , Adolescente , Adulto , Niño , Biología Computacional , Femenino , Humanos , Hipoglucemiantes/farmacología , Inmunoterapia/métodos , Células Secretoras de Insulina/metabolismo , Masculino , Adulto JovenRESUMEN
OBJECTIVE: To better define the rare adverse event (AE) of diabetes mellitus associated with immune checkpoint inhibitors (ICIs). DESIGN AND METHODS: We report the case of a lung cancer patient with diabetic ketoacidosis (DKA) and autoimmune thyroiditis during pembrolizumab treatment. We provide a systematic review of all published cases (PubMed/Web of Science/Cochrane, through November 2018) of autoimmune diabetes mellitus related to blockade of the cytotoxic T-lymphocyte antigen 4 (CTLA-4)-, programmed cell death 1 (PD-1) receptor or its ligand (PD-L1) or combination (ICI) therapy. RESULTS: Our literature search identified 90 patient cases (our case excluded). Most patients were treated with anti-PD-1 or anti-PD-L1 as monotherapy (79%) or in combination with CTLA-4 blockade (15%). On average, diabetes mellitus was diagnosed after 4.5 cycles; earlier for combination ICI at 2.7 cycles. Early-onset diabetes mellitus (after one or two cycles) was observed during all treatment regimens. Diabetic ketoacidosis was present in 71%, while elevated lipase levels were detected in 52% (13/25). Islet autoantibodies were positive in 53% of patients with a predominance of glutamic acid decarboxylase antibodies. Susceptible HLA genotypes were present in 65% (mostly DR4). Thyroid dysfunction was the most frequent other endocrine AE at 24% incidence in this patient population. CONCLUSION: ICI-related diabetes mellitus is a rare but often life-threatening metabolic urgency of which health-care professionals and patients should be aware. Close monitoring of blood glucose and prompt endocrine investigation in case of hyperglycemia is advisable. Predisposing factors such as HLA genotype might explain why some individuals are at risk.
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Antineoplásicos Inmunológicos/efectos adversos , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/diagnóstico , Antineoplásicos Inmunológicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Diabetes Mellitus Tipo 1/sangre , Humanos , Factores Inmunológicos/efectos adversos , Factores Inmunológicos/uso terapéutico , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Persona de Mediana EdadRESUMEN
Aim: Several biomarkers have been proposed to detect pancreatic ß cell destruction in vivo but so far have not been compared for sensitivity and significance. Methods: We used islet transplantation as a model to compare plasma concentrations of miR-375, 65-kDa subunit of glutamate decarboxylase (GAD65), and unmethylated insulin DNA, measured at subpicomolar sensitivity, and study their discharge kinetics, power for outcome prediction, and detection of graft loss during follow-up. Results: At 60 minutes after transplantation, GAD65 and miR-375 consistently showed near-equimolar and correlated increases proportional to the number of implanted ß cells. GAD65 and miR-375 showed comparable power to predict poor graft outcome at 2 months, with areas under the curve of 0.833 and 0.771, respectively (P = 0.53). Using receiver operating characteristic analysis, we defined likelihood ratios (LRs) for rationally selected result intervals. In GADA-negative recipients (n = 28), GAD65 <4.5 pmol/L (LR = 0.15) and >12.2 pmol/L (LR = ∞) predicted good and poor outcomes, respectively. miR-375 could be used in all recipients irrespective of GAD65 autoantibody status (n = 46), with levels <1.4 pmol/L (LR = 0.14) or >7.6 pmol/L (LR = 9.53) as dual thresholds. The posttransplant surge of unmethylated insulin DNA was inconsistent and unrelated to outcome. Combined measurement of these three biomarkers was also tested as liquid biopsy for ß cell death during 2-month follow-up; incidental surges of GAD65, miR-375, and (un)methylated insulin DNA, alone or combined, were confidently detected but could not be related to outcome. Conclusions: GAD65 and miR-375 performed equally well in quantifying early graft destruction and predicting graft outcome, outperforming unmethylated insulin DNA.
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Diabetes Mellitus Tipo 1/cirugía , Glutamato Descarboxilasa/sangre , Rechazo de Injerto/diagnóstico , Insulina/sangre , Trasplante de Islotes Pancreáticos/efectos adversos , MicroARNs/sangre , Adulto , Biomarcadores , Metilación de ADN , Estudios de Seguimiento , Rechazo de Injerto/sangre , Humanos , Insulina/genética , Persona de Mediana Edad , Periodo Posoperatorio , PronósticoRESUMEN
AIMS/HYPOTHESIS: HLA-A*24 carriership hampers achievement of insulin independence in islet allograft recipients. However, less than half of those who fail to achieve insulin independence carry the allele. We investigated whether genetic polymorphism at the recipients' zinc transporter 8-encoding SLC30A8 gene (rs13266634) could complement their HLA-A*24 status in predicting functional graft outcome. METHODS: We retrospectively analysed data of a hospital-based patient cohort followed for 18 months post transplantation. Forty C-peptide-negative type 1 diabetic individuals who received >2 million beta cells (>4000 islet equivalents) per kg body weight in one or two intraportal implantations under similar immunosuppression were genotyped for SLC30A8. Outcome measurements included achievement and maintenance of graft function. Metabolic benefit was defined as <25% CV of fasting glycaemia in the presence of >331 pmol/l C-peptide, in addition to achievement of insulin independence and maintenance of C-peptide positivity. RESULTS: In multivariate analysis, HLA-A*24 positivity, presence of SLC30A8 CT or TT genotypes and BMI more than or equal to the group median (23.9 kg/m2) were independently associated with failure to achieve insulin independence (p = 0.015-0.046). The risk increased with the number of factors present (p < 0.001). High BMI interacted with SLC30A8 T allele carriership to independently predict difficulty in achieving graft function with metabolic benefit (p = 0.015). Maintenance of C-peptide positivity was mainly associated with older age at the time of implantation. Only HLA-A*24 carriership independently predicted failure to maintain acceptable graft function once achieved (p = 0.012). CONCLUSIONS/INTERPRETATION: HLA-A*24, the SLC30A8 T allele and high BMI are associated with poor graft outcome and should be considered in the interpretation of future transplantation trials. TRIAL REGISTRATION: ClinicalTrials.gov NCT00798785 and NCT00623610.
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Glucemia/metabolismo , Índice de Masa Corporal , Diabetes Mellitus Tipo 1/cirugía , Antígeno HLA-A24/genética , Células Secretoras de Insulina/trasplante , Trasplante de Islotes Pancreáticos/efectos adversos , Polimorfismo Genético , Transportador 8 de Zinc/genética , Aloinjertos , Biomarcadores/sangre , Glucemia/efectos de los fármacos , Ensayos Clínicos como Asunto , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Antígeno HLA-A24/inmunología , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Células Secretoras de Insulina/inmunología , Células Secretoras de Insulina/metabolismo , Masculino , Persona de Mediana Edad , Recuperación de la Función , Estudios Retrospectivos , Factores de Riesgo , Resultado del TratamientoRESUMEN
OBJECTIVE: We investigated the effect of HLA class I risk alleles on disease progression in various phases of subclinical islet autoimmunity in first-degree relatives of patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: A registry-based group of siblings/offspring (aged 0-39 years) was monitored from single- to multiple-autoantibody positivity (n = 267) and from multiple-autoantibody positivity to clinical onset (n = 252) according to HLA-DQ, -A*24, -B*18, and -B*39 status. Genetic markers were determined by PCR sequence-specific oligotyping. RESULTS: Unlike HLA-B*18 or -B*39, HLA-A*24 was associated with delayed progression from single- to multiple-autoantibody positivity (P = 0.009) but not to type 1 diabetes. This occurred independently from older age (P < 0.001) and absence of HLA-DQ2/DQ8 or -DQ8 (P < 0.001 and P = 0.003, respectively), and only in the presence of GAD autoantibodies. In contrast, HLA-A*24 was associated with accelerated progression from multiple-autoantibody positivity to clinical onset (P = 0.006), but its effects were restricted to HLA-DQ8+ relatives with IA-2 or zinc transporter 8 autoantibodies (P = 0.002). HLA-B*18, but not -B*39, was also associated with more rapid progression, but only in HLA-DQ2 carriers with double positivity for GAD and insulin autoantibodies (P = 0.004). CONCLUSIONS: HLA-A*24 predisposes to a delayed antigen spreading of humoral autoimmunity, whereas HLA-A*24 and -B*18 are associated with accelerated progression of advanced subclinical autoimmunity in distinct risk groups. The relation of these alleles to the underlying disease process requires further investigation. Their typing should be relevant for the preparation and interpretation of observational and interventional studies in asymptomatic type 1 diabetes.
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Autoanticuerpos/sangre , Autoinmunidad/genética , Diabetes Mellitus Tipo 1/patología , Antígeno HLA-A24/genética , Antígeno HLA-B18/genética , Antígenos HLA-DQ/genética , Islotes Pancreáticos/inmunología , Adolescente , Adulto , Niño , Preescolar , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Progresión de la Enfermedad , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Lactante , Recién Nacido , Anticuerpos Insulínicos/sangre , Masculino , Sistema de Registros , Factores de Riesgo , Adulto JovenRESUMEN
A disproportional increase of circulating GAD65 within hours from an intraportal islet allotransplantation has been validated as biomarker of beta cell loss and poor functional outcome. More sensitive assays are, however, needed to allow detection of episodes of subtle beta cell loss during late-stage graft rejection or in the peri-onset period of type 1 diabetes. We applied the same sandwich monoclonal antibody couple reactive towards the C- and N-terminus of GAD65 on three advanced immunoassay platforms-the Cytometric Bead Array (CBA, Becton, Dickinson and Company), ElectroChemiLuminescence ImmunoAssay (ECLIA, Meso Scale Discovery) and digital ELISA technology (Single Molecule Array-SIMOA, Quanterix. We then compared analytical performance (linearity, imprecision, limit of detection and functional sensitivity), correlation of results, and practicality. All evaluated techniques showed linearity up to at least 500 ng/dL (76.9 pmol/L). SIMOA achieved the lowest imprecision. The 3 platforms correlate well with each other and could all detect subpicomolar concentrations of GAD65 in plasma, but only SIMOA and CBA could quantify down to that range. SIMOA can achieve the highest sample throughput. The three methods tested allow sensitive detection of GAD65, but SIMOA appears best suited for automated quantification of subpicomolar concentrations.
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Glutamato Descarboxilasa/análisis , Glutamato Descarboxilasa/sangre , Inmunoensayo/instrumentación , Biomarcadores/sangre , Análisis Químico de la Sangre/instrumentación , Ensayo de Inmunoadsorción Enzimática/instrumentación , Humanos , Proteínas Recombinantes/análisis , Proteínas Recombinantes/sangre , Sensibilidad y EspecificidadRESUMEN
AIMS: Insulin autoantibodies (IAA) are often the first marker of autoimmunity detected in children in the preclinical phase of type 1 diabetes (T1D). Currently, the vast majority of laboratories adopt the radiobinding micro-assay (RBA) for measuring IAA. Our aim was to replace RBA with a novel non-radioactive IAA Luciferase Immuno Precipitation System (LIPS) assay with improved performance. METHODS: We developed (pro)insulin antigens with alternative placements of a NanoLuc™ luciferase reporter (NLuc). Performance in LIPS was evaluated by testing sera from new onset T1D (n = 80), blood donors (n = 123), schoolchildren (n = 186), first-degree relatives (FDRs) from the Bart's Oxford family study (n = 53) and from the Belgian Diabetes Registry (n = 136), coded sera from the Islet Autoantibody Standardization Program (IASP) (T1D n = 50, blood donors n = 90). RESULTS: IAA LIPS based on B chain-NLuc proinsulin or B chain-NLuc insulin, in which NLuc was fused at the C-terminus of the insulin B chain, required only 2 µL of serum and a short incubation time, showed high concordance with RBA (Spearman r = 0.866 and 0.833, respectively), high assay performance (B chain-NLuc proinsulin ROC-AUC = 0.894 and B chain-NLuc insulin ROC-AUC = 0.916), and an adjusted sensitivity at 95% specificity ranking on par with the best assays submitted to the two most recent IASP workshops. In FDRs, the IAA LIPS showed improved discrimination of progressors to T1D compared to RBA. CONCLUSIONS: We established a novel high-performance non-radioactive IAA LIPS that might replace the current gold standard RBA and find wide application in the study of the IAA response in T1D.
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Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/diagnóstico , Colorantes Fluorescentes/metabolismo , Inmunoprecipitación/métodos , Anticuerpos Insulínicos/análisis , Luciferasas/metabolismo , Autoanticuerpos/sangre , Biomarcadores/análisis , Biomarcadores/metabolismo , Estudios de Casos y Controles , Niño , Colorantes Fluorescentes/análisis , Células HEK293 , Humanos , Insulina/inmunología , Anticuerpos Insulínicos/sangre , Valor Predictivo de las Pruebas , Pronóstico , Proinsulina/inmunología , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: We investigated whether islet autoantibody profile, HLA-DQ genotype, and age influenced a 20-year progression to diabetes from first autoantibody positivity (autoAb+) in first-degree relatives of patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: Persistently islet autoAb+ siblings and offspring (n = 462) under 40 years of age were followed by the Belgian Diabetes Registry. AutoAbs against insulin (IAA), GAD (GADA), IA-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A) were determined by radiobinding assay. RESULTS: The 20-year progression rate of multiple-autoAb+ relatives (n = 194) was higher than that for single-autoAb+ participants (n = 268) (88% vs. 54%; P < 0.001). Relatives positive for IAA and GADA (n = 54) progressed more slowly than double-autoAb+ individuals carrying IA-2A and/or ZnT8A (n = 38; P = 0.001). In multiple-autoAb+ relatives, Cox regression analysis identified the presence of IA-2A or ZnT8A as the only independent predictors of more rapid progression to diabetes (P < 0.001); in single-autoAb+ relatives, it identified younger age (P < 0.001), HLA-DQ2/DQ8 genotype (P < 0.001), and IAA (P = 0.028) as independent predictors of seroconversion to multiple positivity for autoAbs. In time-dependent Cox regression, younger age (P = 0.042), HLA-DQ2/DQ8 genotype (P = 0.009), and the development of additional autoAbs (P = 0.012) were associated with more rapid progression to diabetes. CONCLUSIONS: In single-autoAb+ relatives, the time to multiple-autoAb positivity increases with age and the absence of IAA and HLA-DQ2/DQ8 genotype. The majority of multiple-autoAb+ individuals progress to diabetes within 20 years; this occurs more rapidly in the presence of IA-2A or ZnT8A, regardless of age, HLA-DQ genotype, and number of autoAbs. These data may help to refine the risk stratification of presymptomatic type 1 diabetes.
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Autoanticuerpos/sangre , Diabetes Mellitus Tipo 1/sangre , Progresión de la Enfermedad , Antígenos HLA-DQ/genética , Sistema de Registros , Adolescente , Adulto , Bélgica , Niño , Preescolar , Diabetes Mellitus Tipo 1/genética , Femenino , Estudios de Seguimiento , Humanos , Lactante , Insulina/sangre , Masculino , Modelos de Riesgos Proporcionales , Factores de Riesgo , Encuestas y Cuestionarios , Adulto Joven , Transportador 8 de Zinc/sangreRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0166702.].
RESUMEN
BACKGROUND: The hyperglycemic clamp test, the gold standard of beta cell function, predicts impending type 1 diabetes in islet autoantibody-positive individuals, but the latter may benefit from less invasive function tests such as the proinsulin:C-peptide ratio (PI:C). The present study aims to optimize precision of PI:C measurements by automating a dual-label trefoil-type time-resolved fluorescence immunoassay (TT-TRFIA), and to compare its diagnostic performance for predicting type 1 diabetes with that of clamp-derived C-peptide release. METHODS: Between-day imprecision (n = 20) and split-sample analysis (n = 95) were used to compare TT-TRFIA (AutoDelfia, Perkin-Elmer) with separate methods for proinsulin (in-house TRFIA) and C-peptide (Elecsys, Roche). High-risk multiple autoantibody-positive first-degree relatives (n = 49; age 5-39) were tested for fasting PI:C, HOMA2-IR and hyperglycemic clamp and followed for 20-57 months (interquartile range). RESULTS: TT-TRFIA values for proinsulin, C-peptide and PI:C correlated significantly (r2 = 0.96-0.99; P<0.001) with results obtained with separate methods. TT-TRFIA achieved better between-day %CV for PI:C at three different levels (4.5-7.1 vs 6.7-9.5 for separate methods). In high-risk relatives fasting PI:C was significantly and inversely correlated (rs = -0.596; P<0.001) with first-phase C-peptide release during clamp (also with second phase release, only available for age 12-39 years; n = 31), but only after normalization for HOMA2-IR. In ROC- and Cox regression analysis, HOMA2-IR-corrected PI:C predicted 2-year progression to diabetes equally well as clamp-derived C-peptide release. CONCLUSIONS: The reproducibility of PI:C benefits from the automated simultaneous determination of both hormones. HOMA2-IR-corrected PI:C may serve as a minimally invasive alternative to the more tedious hyperglycemic clamp test.
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Péptido C/sangre , Diabetes Mellitus Tipo 1/sangre , Hiperglucemia/sangre , Proinsulina/sangre , Adolescente , Adulto , Autoanticuerpos/sangre , Niño , Preescolar , Diabetes Mellitus Tipo 1/patología , Ayuno , Femenino , Prueba de Tolerancia a la Glucosa , Hemoglobina Glucada/metabolismo , Humanos , Hiperglucemia/patología , Insulina/sangre , Resistencia a la Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Pronóstico , Análisis de RegresiónRESUMEN
OBJECTIVES: We evaluated the Bio-Rad (Irvine, CA) D-100 and the Sebia (Lisses, France) Capillarys 3 Tera for the measurement of hemoglobin A1c (HbA1c) in venous blood samples. METHODS: Whole-blood samples and control material were analyzed with the D-100 and Capillarys 3 Tera and compared with our routine method, HLC-723G7 (Tosoh, Tokyo, Japan). An evaluation protocol to test precision, trueness, linearity, carryover, and selectivity was set up according to Clinical and Laboratory Standards Institute guidelines. The results were presented in National Glycohemoglobin Standardization Program and International Federation of Clinical Chemistry (IFCC) units. RESULTS: Both systems showed excellent precision (total coefficients of variation <2%, IFCC) and bias (<0.3% or 3 mmol/mol). Linearity was demonstrated for HbA1c values from 3.8% (18 mmol/mol) to 18.5% (179 mmol/mol). Results were correlated with the routine method using Bland-Altman analysis, showing a mean difference of 0.33% or 3.6 mmol/mol for the D-100 and of 0.25% or 2.6 mmol/mol for the Capillarys 3 Tera vs HLC-723G7. None of the automated instruments were prone to interferences by labile HbA1c (≤10 g/L glucose), carbamylated hemoglobin (≤0.5 mmol/L potassium cyanate), hemoglobin variants, bilirubin (≤15 mg/dL), and triglycerides (≤3,360 mg/dL). CONCLUSIONS: The Bio-Rad D-100 and the Sebia Capillarys 3 Tera instruments performed well for the determination of HbA1c in terms of quality criteria as well as for sample throughput.
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Hemoglobina Glucada/análisis , Hemoglobinometría/instrumentación , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Hemoglobinometría/métodos , HumanosRESUMEN
OBJECTIVE: We investigated whether changes in islet autoantibody profile and presence of HLA risk markers, reported to predict rapid ß-cell loss in pre-type 1 diabetes, associate with poor functional outcome in islet allograft recipients. RESEARCH DESIGN AND METHODS: Forty-one patients received ≥2.3 million ß-cells/kg body wt in one to two intraportal implantations. Outcome after 6-18 months was assessed by C-peptide (random and stimulated), insulin dose, and HbA1c. RESULTS: Patients carrying HLA-A*24-positive or experiencing a significant autoantibody surge within 6 months after the first transplantation (n = 19) had lower C-peptide levels (P ≤ 0.003) and higher insulin needs (P < 0.001) despite higher HbA1c levels (P ≤ 0.018). They became less often insulin independent (16% vs. 68%, P = 0.002) and remained less often C-peptide positive (47% vs. 100%, P < 0.001) than recipients lacking both risk factors. HLA-A*24 positivity or an autoantibody surge predicted insulin dependence (P = 0.007). CONCLUSIONS: HLA-A*24 and early autoantibody surge after islet implantation associate with poor functional graft outcome.
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Autoanticuerpos/inmunología , Diabetes Mellitus Tipo 1/terapia , Antígeno HLA-A24/genética , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Trasplante de Islotes Pancreáticos , Adulto , Aloinjertos , Péptido C/metabolismo , Proteínas de Transporte de Catión/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Femenino , Glutamato Descarboxilasa/inmunología , Humanos , Células Secretoras de Insulina , Masculino , Persona de Mediana Edad , Trasplante Homólogo , Resultado del Tratamiento , Transportador 8 de ZincRESUMEN
There is a clinical need for plasma tests to detect and quantify the in vivo destruction of pancreatic ß-cells in type 1 diabetes. We previously developed a time-resolved fluorescence immunoassay (TRFIA) to glutamate decarboxylase 65 kDa (GAD65) (GAD65-TRFIA) that was able to detect the synchronous necrotic destruction of transplanted ß-cells in the hours after their infusion in the liver. This GAD65-TRFIA, however, lacked sensitivity to detect continued ß-cell rejection beyond this acute phase. The aim of present study was to gain at least an order of magnitude in analytical sensitivity by switching to Becton Dickinson cytometric bead array (CBA) (GAD65-CBA) enhanced sensitivity format, using the same couple of monoclonal antibodies. We compared the performances of GAD65-CBA and GAD65-TRFIA using Clinical and Laboratory Standards Institute protocols for linearity, imprecision, specificity, limit of detection, and functional sensitivity. We conducted a method comparison and assessed the biologic potential on samples from human recipients of islet grafts. The GAD65-CBA showed acceptable linearity and imprecision. Switching from TRFIA to CBA lowered functional sensitivity by a factor 35 and lowered limit of detection by a factor 11 with minimal need for method optimization. The enhanced sensitivity greatly expands the application domain of our biomarker and allowed for the first time to detect ongoing ß-cell destruction up to at least 1 day after islet transplantation. We conclude that the GAD65-CBA is suitable for biological and clinical assessment of the real-time destruction of ß-cells in intraportal transplantation.
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Apoptosis , Diabetes Mellitus Tipo 1/sangre , Glutamato Descarboxilasa/sangre , Células Secretoras de Insulina/metabolismo , Trasplante de Islotes Pancreáticos , Biomarcadores/sangre , Diabetes Mellitus Tipo 1/terapia , Fluoroinmunoensayo/métodos , Humanos , Inmunoensayo/métodos , MicroesferasRESUMEN
AIMS/HYPOTHESIS: We examined whether measures of glycaemic variability (GV), assessed by continuous glucose monitoring (CGM) and self-monitoring of blood glucose (SMBG), can complement or replace measures of beta cell function and insulin action in detecting the progression of preclinical disease to type 1 diabetes. METHODS: Twenty-two autoantibody-positive (autoAb(+)) first-degree relatives (FDRs) of patients with type 1 diabetes who were themselves at high 5-year risk (50%) for type 1 diabetes underwent CGM, a hyperglycaemic clamp test and OGTT, and were followed for up to 31 months. Clamp variables were used to estimate beta cell function (first-phase [AUC5-10 min] and second-phase [AUC120-150 min] C-peptide release) combined with insulin resistance (glucose disposal rate; M 120-150 min). Age-matched healthy volunteers (n = 20) and individuals with recent-onset type 1 diabetes (n = 9) served as control groups. RESULTS: In autoAb(+) FDRs, M 120-150 min below the 10th percentile (P10) of controls achieved 86% diagnostic efficiency in discriminating between normoglycaemic FDRs and individuals with (impending) dysglycaemia. M 120-150 min outperformed AUC5-10 min and AUC120-150 min C-peptide below P10 of controls, which were only 59-68% effective. Among GV variables, CGM above the reference range was better at detecting (impending) dysglycaemia than elevated SMBG (77-82% vs 73% efficiency). Combined CGM measures were equally efficient as M 120-150 min (86%). Daytime GV variables were inversely correlated with clamp variables, and more strongly with M 120-150 min than with AUC5-10 min or AUC120-150 min C-peptide. CONCLUSIONS/INTERPRETATION: CGM-derived GV and the glucose disposal rate, reflecting both insulin secretion and action, outperformed SMBG and first- or second-phase AUC C-peptide in identifying FDRs with (impending) dysglycaemia or diabetes. Our results indicate the feasibility of developing minimally invasive CGM-based criteria for close metabolic monitoring and as outcome measures in trials.