[Obesity and risk factors for metabolic syndrome in young people with type 1 diabetes]. / Obesidad y factores de riesgo del síndrome metabólico en jóvenes con diabetes tipo 1.

Changes in the clinical presentation of diabetes mellitus in childhood and adolescence associated with obesity have resulted in an overlap of the two most common types of diabetes with a greater clinical heterogeneity. In order to characterize the type of diabetes at onset and assess the effect of obesity, 50 children with diabetes were studied. The patients were divided into two groups according to their nutritional status at diagnosis (over-weight/obese vs. normal weight). Insulin reserve was evaluated by measuring basal C-peptide and stimulated C-peptide in response to a mixed meal (MMTT) as well as HLA-DQB1 genotype, antibodies, and family history of risk factors for metabolic disease. Of all 50 patients, 38% was overweight/obese, 84% had a positive family history of metabolic syndrome, 82% had positive antibodies, and 100% were positive for the high-risk HLA-DQB1 genotype. No significant differences were found in fasting C-peptide or glycemic index/C-peptide levels between the two groups. In the overweight/obese group C-peptide response to MMTT showed higher levels at 60 and 120 minutes (p = 0.02 and 0.03) and the area under the curve for C-peptide was also higher (1.77 ng / ml vs. 5.5 ng/ ml, p = 0.0007) than in the normal-weight group. In conclusion, overweight/obese patients with type 1A diabetes had a greater pancreatic reserve, suggesting that nutritional status may accelerate disease onset.

Frequency of pancreatic beta-cell autoimmunity markers in patients with autoimmune thyroid disease.

A total of 305 ambulatory patients recruited at the Division of Endocrinology, Hospital de Clínicas, University of Buenos Aires, with autoimmune thyroid disease (AITD) were studied to search for associations between autoimmune thyroid disease and presence of serum markers of autoimmune diabetes mellitus. Screening for markers of pancreatic beta-cell autoimmunity was performed by radioligand binding assays (RBA) as follows: autoantibodies to glutamic acid decarboxylase (GADA) and proinsulin (PAA) were determined in all sera, whereas autoantibodies to protein tyrosine phosphatase (IA-2A) and insulin (IAA) were additionally measured in 200 sera randomly selected from the total collection. In addition, every GADA positive serum among the remaining 105 sera was systematically tested for the presence of IA-2A and IAA. In the cohort of 305 AITD patients 22 (7.2%) were previously diagnosed as type 1, type 2 or insulin-requiring type 2 diabetics. Ten of these patients presented serum marker positivity specific for beta-cell autoantigens and 12 were marker negative. On the other hand, considering the majority of non-diabetic AITD patients (n = 283), beta3-cell marker positivity was detected in 17 individuals (6.0%). The prevalence of autoimmune diabetes markers was much higher in the studied population than in the general population utilized as a control group, and GADA was the most frequent marker.

Detection and immunochemical characterization of glutamic acid decarboxylase autoantibodies in patients with autoimmune diabetes mellitus.

Since GAD65 undergoes post-translational processing and targeting to subcellular compartments and membranes, it may exhibit different immunochemical properties in the cell context compared with the soluble protein expressed in the cell-free eukaryotic system used in the reference method for GADA assessment (radioligand binding assay (RBA)). In the present work, we detected and characterized GADA in 72 sera from patients with type 1 diabetes mellitus (DM) and 14 sera from adult-onset diabetes patients using analytical systems in which GAD65 is expressed in a cellular context: confocal indirect immunofluorescence (IIF) and electron microscopy after immunogold labeling on monolayers of transfected Chinese hamster ovary (CHO) cells, and immunoprecipitation (IP) of metabolically labeled GAD65. Eighteen serum samples, 16 from type 1 diabetes patients and two from adult-onset diabetes patients, were positive by confocal IIF but scored negative by RBA. All of these 18 sera immunoprecipitated a 65 kDa protein, supporting the existence of the GADA marker in such patients. It may be concluded that GADA negativity by the conventional RBA method using the soluble antigen, as well as negativity for other common markers measured by similar methods, is not enough to rule out the existence of the specific autoimmune component in childhood or adult-onset diabetes. Other analytical methods based in a more physiological presentation of the autoantigen structure, as confocal IIF and IP, bring an extra support to assess the complete repertoire of specific autoantibodies to native-like and membrane-bound, or denatured, beta-cell antigens.

Frequency Of Pancreatic Beta-Cell Autoimmunity Markers In Patients With Autoimmune Thyroid Disease / Frecuencia de marcadores de autoinmunidad beta pancreática en pacientes con enfermedad tiroidea autoinmune

A total of 305 ambulatory patients recruited at the Division of Endocrinology, Hospital de Clínicas, University of Buenos Aires, with autoimmune thyroid disease (AITD) were studied to search for associations between autoimmune thyroid disease and presence of serum markers of autoimmune diabetes mellitus. Screening for markers of pancreatic beta-cell autoimmunity was performed by radioligand binding assays (RBA) as follows: autoantibodies to glutamic acid decarboxylase (GADA) and proinsulin (PAA) were determined in all sera, whereas autoantibodies to protein tyrosine phosphatase (IA-2A) and insulin (IAA) were additionally measured in 200 sera randomly selected from the total collection. In addition, every GADA positive serum among the remaining 105 sera was systematically tested for the presence of IA-2A and IAA. In the cohort of 305 AITD patients 22 (7.2%) were previously diagnosed as type 1, type 2 or insulin-requiring type 2 diabetics. Ten of these patients presented serum marker positivity specific for β-cell autoantigens and 12 were marker negative. On the other hand, considering the majority of non-diabetic AITD patients (n=283), β-cell marker positivity was detected in 17 individuals (6.0%). The prevalence of autoimmune diabetes markers was much higher in the studied population than in the general population utilized as a control group, and GADA was the most frequent marker.

Se investigó la asociación entre enfermedad tiroidea autoinmune y la presencia de marcadores séricos de diabetes mellitus en 305 pacientes ambulatorios con enfermedad tiroidea autoinmune reclutados en la División Endocrinología. La búsqueda de marcadores de autoinmunidad contra las células beta pancreáticas se realizó por la técnica de unión de radioligandos (RBA) como se detalla a continuación: se determinaron autoanticuerpos contra la decarboxilasa del ácido glutámico (GADA) y proinsulina (PAA) en todos los sueros, mientras que los anticuerpos contra la proteína tirosina fosfatasa (IA-2A) e insulina (IAA) fueron medidos en 200 de estos sueros tomados al azar de la colección total. Además, en los restantes 105 pacientes, la presencia de IA-2A y IAA fue evaluada en todos los sueros positivos para GADA. Del grupo de 305 pacientes con enfermedad tiroidea autoinmune 22 (7.2%) fueron diagnosticados previamente como diabéticos tipo 1, tipo 2 o tipo 2 insulino-requirientes. Diez de ellos presentaron positividad para marcadores específicos de autoantígenos de célula β, en tanto 12 fueron negativos. Por otra parte, en 17 de los 283 pacientes (6.0%) con enfermedad tiroidea autoimmune y sin diagnóstico previo de diabetes, se detectó positividad para marcadores de célula β. La prevalencia de marcadores de autoinmunidad asociados a diabetes fue mayor en la población estudiada que en la población general usada como grupo control, siendo GADA el marcador más frecuente.

Development of 2 alternative enzyme-linked immunosorbent assays for routine screening of glutamic acid decarboxylase autoantibodies.

BACKGROUND: Antibodies to GAD65 (GADA) are considered highly predictive humoral markers of the type 1 diabetes mellitus and also of the insulin requirement in adult-onset patients presumptively classified as type 2 diabetics or LADA. METHODS: We present 2 methods for GADA assessment. The first one (fluid phase, ELISA Protocol A) is carried out in a 2-step procedure in which serum GADA are first allowed to react with a fixed dose of GAD65-biotin in solution and the residual free antigen is later assayed by a conventional ELISA. In the second test (solid phase, ELISA Protocol B) GADA are measured in an ELISA that depends on the ability of divalent autoantibodies to form a bridge between immobilized TrxGAD65 and liquid-phase biotinylated TrxGAD65. RESULTS: All normal control samples scored negative in both variants of ELISA and RBA, hence specificity was 100% for all methods; the relative sensitivity of ELISA Protocol A respect of the RBA was 94% and that of ELISA Protocol B was 76%. CONCLUSIONS: Although ELISA Protocol A exhibited a better performance in terms of relative sensitivity than ELISA Protocol B, the simplicity of execution and the intended use of the assay must also be taken in consideration for the final choice.

Cytotoxic T lymphocyte antigen 4 heterozygous codon 49 A/G dimorphism is associated to latent autoimmune diabetes in adults (LADA).

Autoimmune diabetes is an organ specific and multifactorial disorder with a classical onset as insulin dependent diabetes mellitus (IDDM) and with another form of onset as latent autoimmune diabetes in adults (LADA), which has a slower onset and a later progress to insulin dependency as a result of the beta cells destruction. The cytotoxic T lymphocyte-antigen 4 (CTLA4) has been identified as a susceptible marker of the disease; it is considered a down regulator of T cell function, playing a key role in autoimmunity. We analyzed CTLA4 codon 49 A/G polymorphism in 123 IDDM patients, 63 LADA patients and 168 healthy non-diabetic control individuals. The frequency of the heterozygous A/G genotype in LADA patients was significantly increased compared to IDDM patients (55.6 vs. 39.8%, p = 0.0415). There was no statistical significant difference in the distribution of the A/G dimorphism between autoimmune diabetes patients (LADA or IDDM) and non-diabetic control individuals. HLA DQ region is responsible for the genetic susceptibility to autoimmune diabetes in IDDM patients in about 50% and it has a lower effect in genetic susceptibility in LADA patients. Several other genetic loci are needed to develop autoimmune diabetes in adult patients. Therefore, LADA may be the result of a combined minor risk loci effect in a major risk haplotype.

Single-tube test for autoantibodies to glutamic acid decarboxylase and proinsulin as first-line screening for autoimmunity in adult-onset diabetic patients.

The aim of this work was develop a new combined radioligand-binding assay (RBA-combi) for the rapid and simultaneous determination of two autoimmunity markers, GADA and PAA, known to be differentially distributed in young and in some adult diabetic patients. The methodology was applied to sera from 85 young type 1 and 98 adult-onset diabetic patients with different marker profiles and insulin requirements, and to 53 normal control sera. Among type 1 diabetes sera used as autoimmunity controls, 100% of those with at least one positive marker by single methods and 17.7% of those with double negative markers were positive by RBA-combi (RBA-combi+). Among sera from adult-onset diabetes, 100% of those PAA+ (GADA+ or GADA-), 92.3% of GADA+/PAA-, and 1.3% of GADA-/PAA- were RBA-combi+. In conclusion, the new RBA-combi allowed the simultaneous detection of GADA and PAA markers with acceptable performance. Moreover, 16 out of 18 (88.9%) of adult patients RBA-combi+ evolved to insulin requirement, suggesting that this test is a valuable tool for assessing autoimmune processes associated to future impairment of insulin secretion.

Obtención de 35 S-proinsulina humana recombinante y su aplicación en un ensayo radiométrico combinado: Utilidad del método desarrollado para predecir el requerimiento de insulina en pacientes diabéticos adultos / Obtaining of 35 recombinant human S-proinsulina and its application in a combined radiometric test: Utility of the developed method to predict the insulin requirement in adult diabetic patients

Los autoanticuerpos anti glutamano decarboxilasa y anti insulina/proinsulina(GADA e IAA/PAA)tienen valor predictivo del requerimiento insulínico en pacientes diabéticos con comienzo clínico en edad adulta. En este trabajo se desarrolló un nuevo trazador 35 S-Proinsulina y se lo utilizó en un ensayo de unión de radioligando combinado con el trazador 35 S-GAD para la determinación simultñanea de GADA y PAA (RBA-combi). Se aplico el ensayo a 85 pacientes infanto-juveniles con diabetes tipo 1, a 98 pacientes con comienzo clínico en edad adulta y a 53 controles normales. El 100% de los pacientes con diabetes tipo 1 con al menos un marcador positivo, y el 17,7% de los que eran negativos para ambos marcadores, fueron positivos por el RBA-combi(RBA-comi+). El 100% de los pacientes adultos PAA+(GADA+ o GADA-) el 92,3% de los pacientes GADA+/PAA-, y el 1,3% de los pacientes GADA-/PAA-, fueron RBA-combi+. El 88.9% de los pacientes adultos RBA-combi+evolucionaron a requerimiento insulínico. En conclusión, el nuevo trazador 35 S-Proinsulina fue apto para su utilización en el método radiométrico combinado, permitiendo la detección simultánea de los marcadores GADA Y PAA. El RBA- combi es una herramienta valiosa para detectar procesos autoinmunes asociados a un futuro requerimiento insulínico, en pacientes diabéticos adultos.

A radioligand-binding assay for detecting antibodies specific for proinsulin and insulin using 35S-proinsulin.

A new radioligand-binding assay (RBA) is described for the detection of insulin/proinsulin-specific antibodies using 35S-labeled proinsulin produced by a cell-free reticulocyte extract. Direct use of the crude expression product in the RBA was not feasible because the protein failed to fold properly (or had incorrectly paired disulphide bridges) and purification was hindered by interfering by-products. A refolding protocol and a chromatographic procedure were devised that readily allowed production of purified and immunochemically competent 35S-labeled proinsulin. The new RBA was compared with the reference test, in which the tracer was standard 125I-insulin. The analysis included sera from 41 diabetic patients and 25 healthy controls. Twenty-six (63.4%) and 29 (70.7%) patients scored positive by RBA using 35S-PI and 125I-insulin, respectively. The methods showed a satisfactory correlation with r(2)=0.77 and a slope not significantly different from unity (m=1.16+/-0.10; 95% confidence interval). Since the nuclide used in the assay is 35S, the procedure is compatible with standard assays for GADA and IA-2A, and thus may permit combined assays for the major early markers of autoimmune diabetes.