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Muscle weakness in patients with thyrotoxicosis during hypokalemic episodes (thyrotoxic periodic paralysis [TPP]) occurs sporadically and mostly in males. It is treated by infusion or oral supplementation with potassium and with resolution of the thyrotoxicosis state. The clinical features of TPP resemble familial hypokalemic periodic paralysis (hypoKPP), which has been linked to two mutations in the gene encoding the skeletal muscle calcium channel alpha-1 subunit (CACN1AS; Arg528His and Arg1239His) and to the sodium channel alpha-subunit (SCN4A; Arg672His). We screened for the mutations (CACN1AS by polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP]; SCN4A by single-strand conformation polymorphism analysis) described in hypoKPP in 20 unrelated patients with documented episodes of TPP (mean age, 40.0 +/- 12.3 years 19 males). Forty-eight patients with hyperthyroidism resulting from Graves' disease (48.5 +/- 12.3 years; 13 males), 1 patient with idiopathic hypoKPP (a 32-year-old male) and 32 healthy subjects (41.0 +/- 19.1 years; 16 males) were included. We found none of the TPP patients carry CACN1AS and SCN4A mutations. The hyperthyroid patients and control subjects were also negative for the mutations. The patient with idiopathic hypoKPP was genotyped to have the Arg528His mutation. These results suggest that despite close similarities between TPP and hypoKPP, a likely genetic basis for TPP does not involve the same gene mutations associated with hypoKPP.

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BACKGROUND: Circulating antibodies to glutamic acid decarboxylase (GADab) and tyrosine phosphatase-like molecule IA-2 (IA-2ab) are major indicators for auto-immune destruction of pancreatic islet cells. They identify a majority of Caucasians with type 1 diabetes and approximately 50% of Asians, providing evidence of an idiopathic aetiology in the latter. The present study investigated these autoantibodies in a mixed ethnic group. METHODS: Hospital clinic patients with clinically defined type 1 (n = 93) and type 2 (n = 300) diabetes and representing Singapore's major ethnic groups--Chinese, Indians and Malays--were studied. GADab and IA-2ab frequencies, and association of autoimmunity status with clinical and biochemical profiles were analysed. RESULTS: Radio-immunoprecipitation assays detected either or both antibodies (seropositivity) in 41.9% of subjects with type 1 diabetes. GADab was detected in 36.6% and IA-2ab in 23.7% of type 1 diabetics. Prevalence of IA-2ab showed a reduction in frequency with disease duration (P = 0.026). In clinical type 2 diabetics, seropositivity was 10.0% with higher frequency in Malays (17.5%) than Chinese (9.7%) and Indians (4.5%). Multivariate analysis revealed that low fasting C-peptide was associated with seropositivity (odds ratio (OR) = 0.15; 95% confidence interval (CI) = 0.04-0.58). A significant relationship (OR = 13.5; 95% CI = 5.0-36.7) between insulin requirement and duration (>5 years) was also revealed. In patients with type 2 diabetes there was a trend of gradual progression to insulin dependency. However, there was considerable variation in body mass index between ethnic subgroups of type 2 diabetics, particularly for Chinese (mean (SD) = 26.0 (4.7)) and Malays (mean (SD) = 29.2 (5.9); P < 0.001). CONCLUSIONS: Presence of both antibodies in our mixed ethnic group of type 1 diabetes patients was much lower than in Caucasians. Significant numbers of patients were seronegative for antibodies. Influences due to ethnicity and adiposity would require further investigations.

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BACKGROUND AND AIMS: Islet autoantibodies are known markers for type 1 diabetes with an immune-mediated basis; their isotype or subclass profiles may also provide clues to changes in immune response during disease or after intervention. For ICAs and GADab, the IgG1 subclass consistently dominates in recent-onset disease. The aims of our study were to determine the isotype patterns for IA-2ab in Asian Chinese patients with autoimmune diabetes. MATERIALS AND METHODS: From an initial screening of over 400 diabetes patients, 40 subjects (mean age 22.2 +/- 15.8 years) with IA-2ab were enrolled for this study. IA-2ab was detected by radioimmunoassay of [35S]-labelled recombinant human IA-2 ic(605 - 979). Of them, 31 (median age 15 years, range 2 - 57 years; 16 children) had clinical type 1 diabetes (that is, they required insulin at onset or within 1 year) with the majority having been recently diagnosed (< 1 year). The other 9 patients had clinical type 2 diabetes phenotype. RESULTS: IA-2ab IgG subclasses determined with monospecific secondary antibodies showed that both type 1 diabetic adults and children had similarly non-restricted isotype patterns with a strong presence of IgG1-IA-2ab. The rank order was IgG1 > 3 > 2 > 4; 15 subjects had detectable IgG4-IA-2ab. Clonality of immune response determined with kappa/lambda chain-specific antibodies also showed a non-restricted pattern. Patients aged 38.2 +/- 15.2 years with type 2 diabetes had broad patterns of isotypes - IgG1/3 was detected more frequently (n = 8) than IgG2/4 (n = 5). Of three patients on insulin treatment, one was also positive for GADab. The remaining 6 patients were on oral hypoglycaemic treatment. IA-2ab in type 2 diabetes showed a low titre compared to type 1 diabetes. CONCLUSIONS: Isotype responses to IA-2 had a strong IgG1 presence, similar to ICAs and GADab. With IgG3 subclass representation, a predominant Th1 milieu in the systemic environment is likely. There is no suggestion of differences in immune response to IA-2 between adults and children with type 1 diabetes.

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Type 1 diabetes in most Asian populations may not have a salient autoimmune basis when assessed with single determinations of the major markers, islet cell antibodies (ICAs) and glutamic acid decarboxylase antibodies (GAD65ab). With the inclusion of antibodies to tyrosine phosphatase-like protein IA-2 (IA-2ab) as an additional major marker, we re-examined autoimmune diabetes in a group of Chinese patients. We studied 272 subjects at various stages of disease with blood samples procured for biochemical analysis. ICAs were measured by immunofluorescence, GAD65ab and IA-2ab by radioimmunoassay. Sixty-seven patients fulfilled clinical diagnosis of type 1 diabetes and the remaining 205 patients were type 2. Prevalence of single autoantibody type in recent-onset type 1 diabetes ( < 1 year duration; n = 47) showed 10.6% with ICAs, 44.7% GAD65ab and 36.2% IA-2ab. GAD65ab account for more than two-thirds of the markers found in type 1 diabetes. Combined analysis further showed that 51.1% had at least one antibody type, 31.9% with two or more antibodies and 8.5% with all three antibodies. Islet autoimmunity presence in childhood-onset type 1 diabetes improved with the addition of IA-2ab, though less impact was seen in the adult-onset. Similarly, combined analysis for type 2 patients with recent diabetes showed a modest increase to 13% with islet autoimmunity compared to 8% when assessed by GAD65ab alone. Combining IA-2ab and GAD65ab assays results detected slightly more immune-mediated diabetes, compared to using a single GAD65ab determination. Non-autoimmune causes need to be considered in the pathogenesis of type 1 diabetes in Chinese, particularly in adults.

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OBJECTIVE: To determine the prevalence of islet cell antibodies (ICA) and antibodies to glutamic acid decarboxylase (GAD) in Asian children with diabetes mellitus (DM) at the time of diagnosis. PATIENTS AND METHODS: 41 children were studied at their initial presentation from 1993 to 2000. RESULTS: Mean age of onset (+/- SD) of DM was 7.6 (+/- 4.2) years. One or both of the two autoantibodies, ICA and anti-GAD, were present in 17 of the 41 children (41.5%) at the time of diagnosis. Comparing the group of children with autoantibodies and the group without detectable autoantibodies, there were no significant differences in body mass index (15.4 vs. 16.3 kg/m2), age of onset (7.4 vs. 7.8 years), random C-peptide levels (203 vs. 311 pmol/l), HbA1c levels (13.2 vs. 12.7%), and frequency of diabetic ketoacidosis at presentation (53.3 vs. 55%). CONCLUSION: Prevalence of antibodies at presentation of DM in Singapore (41.5%) is lower than in Caucasian populations (60-90%). Other autoantibodies yet to be identified may be contributory. Alternatively, non-immune mediated mechanisms may be responsible for a significant proportion of type 1 DM in Singapore children.

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INTRODUCTION: Microalbuminuria has been established as a marker of incipient diabetic nephropathy, and a regular screening programme has been advocated in patients with diabetes mellitus. We investigated if urine albumin levels normalised by creatinine give results comparable to the urinary albumin excretion rate (UAER) with a timed 24-h urine for detecting microalbuminuria. MATERIALS AND METHODS: Morning urine specimens and 24-h collections were obtained from 65 diabetic patients. Albumin and creatinine levels in the spot urine specimens were measured in a single rapid (7 min) assay format using the Bayer DCA2000+ desktop system. Results of albumin/creatinine ratio and urine albumin concentration were then compared to the reference laboratory method of measuring UAER with the timed 24-h urine sample. RESULTS: The determination of the albumin/creatinine ratio gave good performance characteristics for diagnosis of microalbuminuria (defined as > 30 mg/g creatinine) with sensitivity of 71.4% and specificity of 98.0%. With urine albumin concentration alone, sensitivity was 64.3% and specificity was 96.1%. Receiver operating characteristic (ROC) curves, however, suggest similar diagnostic usefulness for screening microalbuminuria with albumin levels expressed in concentration units or as a ratio of creatinine compared to the reference method. Analysis also indicated that lowering the established cut-off values in general, improves diagnostic performance. CONCLUSIONS: Measurement of microalbuminuria with a spot morning specimen using the DCA2000+ desktop system that simultaneously measures albumin and creatinine levels, provides a rapid and reliable method for incipient diabetic nephropathy in clinical practice.

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It is not clear if a Th1/Th2 imbalance in Type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) would lead to a particular antigen-specific IgG subclass dominant as had been shown in the mouse model. In new-onset Type 1 diabetics, an autoantibody response to glutamate decarboxylase (GADab) is frequently observed but the GADab subclass repertoire is not well-established. We determined the systemic levels of representative Th1 and Th2 cytokines and the GADab IgG subclass distribution in 41 Chinese IDDM patients of whom 26 were recently diagnosed (< or = 1 year) and 32 had GADab, to ascertain a likely association of antigen-specific antibody isotype and the Th1/Th2 dichotomy. With high-sensitivity ELISA systems that measure sub-picogram cytokine concentrations, 26 of the 41 patients (63.4%) had at least one of the pro-inflammatory Th1 cytokines (TNF-alpha, IFN-gamma and IL-12) detected. Fewer patients (4/41) had the anti-inflammatory Th2 cytokine IL-4 detected. For IL-10, all subjects had measurable quantities but only three diabetics had levels above the upper limit for healthy subjects (n = 20). Grouped according to the profile of detectable cytokines, there were 24 Th1, 2 Th2 and 2 Th0 patterns. GAD-specific IgG1 antibody was more frequently expressed; 22 of 32 GADab[+] patients. The rank order for the GADab subclasses was IgG1 > 4 > 3 > 2; IgG2 was found in 11 GADab[+] patients. Recent-onset diabetics have a similar ranking of the GAD-specific IgG subclasses. In human Type 1 diabetes, a predominance of GAD-specific IgG1 antibody response is observed together with a dominant Th1 cytokine pattern.

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AIM: Leptin levels in the overweight and differences between men and women may be confounded by sex hormones in obesity-related type 2 diabetes. We carried out a study of leptin and testosterone and the effect of diabetes on this relationship in 71 Chinese subjects (51 with type 2 diabetes and 20 healthy non-diabetics) of whom 32 were overweight (body mass index (b.m.i.) > 25.0 kg/m2). METHODS AND RESULTS: Leptin levels were similar in the diabetic (median 7.4, interquartile range 4.2-10.6 ng ml-1) and non-diabetic (11.3 (6.0-16.3) ng ml-1) subjects. Testosterone and free testosterone in diabetics and non-diabetics were similar for both gender. Regardless of diabetes status, testosterone and its free form tend to increase in overweight women and decrease in men. Leptin correlated with testosterone in women with diabetes (Pearson correlation coefficient r = 0.495, p = 0.037) but not in diabetic men. Sixty-eight per cent of leptin variance was affected by b.m.i, insulin, sex (female) and diabetes status in a regression model that excluded testosterone (total and free). However, testosterone and insulin were predictors of leptin changes in the non-diabetic subjects. In diabetes, b.m.i., sex (female) and insulin remained as predictors of leptin changes. CONCLUSIONS: These results suggest that apart from body adiposity, testosterone also influences leptin levels and that diabetes has a significant effect on this association.

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BACKGROUND: Microalbuminuria is an early marker of prognostic significance in diabetic renal disease. However, testing for microalbuminuria in a timed sample of urine using the double antibody radioimmunoassay (RIA) method is cumbersome and requires special laboratory facilities. Recently, a test strip for microalbuminuria, the Micral Test was available and we evaluated the performance of this test strip as a screening method for detection of microalbuminuria. METHODS: One hundred consecutive diabetic patients who were tested to be dipstick-negative (Albustix) for proteinuria were enrolled for the study. Micral Tests were performed on a paired first morning and random urine specimen from the same patient and the results compared with a timed 24-hour urine measurement of urine albumin excretion using the RIA method. RESULTS: Eighteen specimens were tested positive by the RIA method with a urinary albumin range of 32-177 mg/24 hours. With the Micral Test, the following sensitivity, specificity, positive and negative predictive values were obtained: 66.7%. 97.6%, 85.7% and 93.0% for the first morning urine specimens, and 77.8%, 91.5%, 66.7% and 94.9% for the random urine specimens. CONCLUSIONS: These results suggest that Micral Test with either the first morning or random urine specimen offers a simple, reliable, rapid and convenient method for screening of microalbuminuria in the diabetic patient.

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An autoimmune basis for the pathogenesis of insulin-dependent diabetes mellitus (IDDM) is supported by the frequent presence of autoantibodies - islet cell antibodies (ICAs) and GAD antibodies (GADab). However, in Chinese patients with clinical IDDM, a low prevalence of ICAs was observed. In non-insulin-dependent diabetic (NIDDM) patients, it has been suggested that the presence of GADab may identify a subset of latent autoimmune diabetes in adults (LADA). We determined the frequency of GADab in a large group of 134 IDDM and 168 NIDDM Chinese patients, and assessed the relation with ICAs status. Results showed that 39.6% IDDM and 16.1% NIDDM patients had GADab, and 20.1% and 4.8%, respectively had detectable ICAs. Frequency of GADab positivity was not influenced by whether the patients had youth or adult-onset IDDM or NIDDM, or by duration of diabetes. NIDDM patients seropositive for GADab shared similar clinical characteristics and fasting C-peptide levels with those who were GADab negative. Presence of GADab therefore did not serve to identify a subgroup of patients with latent or slow-onset IDDM. Half (53%) of our IDDM patients had neither GADab nor ICAs. The reason for this observation is unclear. One theory is that other autoantigens yet to be identified may be contributory. Alternatively, in the Chinese, autoimmunity may not be the major factor in the pathogenesis of IDDM.

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Forty-seven patients with newly diagnosed diabetes were followed up for 1 year with lipid parameters (total cholesterol, low-density lipoprotein [LDL] cholesterol, high-density lipoprotein [HDL] cholesterol and triglycerides) and glycated haemoglobin measured at diagnosis and 6-monthly intervals. The prevalence of these lipid parameters exceeding the risk level for coronary heart disease at diagnosis ranged from 34% to 64%, the commonest dyslipidaemic pattern being hypercholesterolaemia (43%), followed by mixed hyperlipidaemia (17%) and hypertriglyceridaemia (17%). There was no improvement in the lipids profile over a 1-year period despite achieving good glycaemic control in both the insulin-dependent diabetes mellitus (IDDM) and non-insulin-dependent diabetes mellitus (NIDDM) patients. Twenty-four (51%) patients will need lipid lowering treatment because of persistent dyslipidaemia despite adequate non-pharmacologic measures. We suggest that an active strategy of early detection and drug treatment for dyslipidaemia is needed for patients with diabetes mellitus.

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Markers of cell-mediated immune activation were studied in 32 Chinese patients with recent-onset insulin-dependent diabetes mellitus (IDDM) as compared with 12 patients with recent-onset non-insulin-dependent diabetes mellitus (NIDDM) and 34 normal subjects. Sera were assessed for soluble markers of T-cell activation (sCD4, sCD8, sIL-2R); the cytokines (IL-1 beta, TNF-alpha, IL-2, IL-6), and T-cell subsets were also determined. Only 1 of the 32 IDDM patients had increased sCD4 levels, 5 had increased sCD8, and 3 had increased sIL-2R. None of the sera from NIDDM patients and control subjects showed such increased levels of soluble markers. Three IDDM patients had detectable IL-1 beta and this weakly so (< 3.5 pg/ml). However, the other cytokine data and the frequency of activated T-cells, CD4+, CD8+ T-cell subsets and CD4:CD8 ratio showed no significant differences among the IDDM, NIDDM and normal subjects. Our data suggest that in addition to a low frequency of islet cell antibodies, Chinese patients with recent onset IDDM also showed a lack of serum markers of cellular activation.

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A new desktop monoclonal antibody-based test system (Bayer Diagnostics DCA 2000) for quantitation of glycated haemoglobin HbA1c is described and evaluated. This method involves a monoclonal antibody to HbA1c which defines its specificity. It requires 9 minutes to complete, and shows good intra- and inter-run precisions (1.3%-3.7%), at 5 HbA1c levels tested--4.1%, 5.7%, 6.1%, 9.2% and 12.4%. Results from 81 blood samples obtained from diabetic patients (3.9%-13.2% HbA1c) showed excellent correlation with a laboratory-based ion-exchange HPLC technique (y = 1.03 [Lab] + 0.103%; Pearson coefficient, r = 0.99). The test can be performed either with a capillary fingerprick or venous blood sample. Only 1 microliter of blood volume is required. Comparison of HbA1c levels of 43 paired capillary and venous samples showed excellent correlation (y = 1.00 [venous] + 0.042%; r = 0.99). The HbA1c values obtained from a cohort of 37 healthy adults, mean (+/- SD) age 33 +/- 9.01 years, gave a value of 5.5 +/- 0.42%. The calculated 95% confidence limits are 4.7%-6.3%. This quick method provides 'stat' HbA1c results, which were hitherto not possible with the laboratory-based methods.

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Insulin autoantibodies (IAA) in patients with autoimmune thyroid disease (n = 97) were determined by a displacement-based ELISA technique. There were 78 patients with Graves' disease and 19 with autoimmune thyroiditis. The assay precision unit, standard deviation score, was used to assess the presence of IAA. It was determined that 7.2% were assessed to be IAA-positive. None of the patients had islet cell antibodies. Thyrotrophin receptor antibodies (TRAb) were only present in patients with Graves' disease. The mean TRAb levels in patients who relapsed (95.0 +/- 141 IU/l, n = 36) were significantly higher (p = 0.042) than those newly diagnosed for Graves' disease (38.3 +/- 36 IU/l, n = 28). Insulin autoantibody levels in these two subgroups were not different from each other. In addition, TRAb levels were not related to IAA levels. A retrospective study of 12 patients on carbimazole treatment indicated that modulation of TRAb levels had no concomitant effect on the development of IAA.

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Immunoprecipitation assays for antibodies to the nicotinic acetylcholine receptor are a specific test for the diagnosis of myasthenia gravis. However, testing has largely been confined to reference laboratories because the preparation of the reagents is laborious, making it less accessible to practising clinicians locally. We recently acquired a test kit produced by RSR Ltd (Avenue Park, Pentwyn, Cardiff) which makes testing of serum samples practicable in smaller centres. We describe our experience in establishing normal and control values in 30 separate samples. We also assayed the sera of ten patients with thyroid disease, some of whom had ophthalmopathy and/or weakness due to periodic paralysis, and none of these tested positive. Three additional patients with thyroid disease and concurrent myasthenia gravis were also inadvertently included in the latter assay and two had high titres and one had equivocal titres. The assay is now run regularly every quarter of the year.

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Human Leucocyte Antigen (HLA) A, B, C, DR, DQ and DP locus alleles in 35 Chinese patients with Graves' disease and 80 controls were typed for using serological and DNA polymerase chain reaction/sequence specific oligonucleotide (PCR/SSO) methods. B46 was found to be strongly associated with male patients (p = 0.0002; RR = 8.2, 95% confidence interval 2.1-32.2). In HLA class 1 alleles, besides B46, the frequencies of A2 and Cw1 were increased and A24 and B63 decreased in patients compared to controls. In HLA class 2 alleles, the frequencies of DRB1*1602, *0301, *1405, DRB5*02, DQB1*0502 were increased and DRB1*1501, DQB1*0301 decreased in patients.

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Islet cell antibodies (ICAs) in Chinese (23 IDDM, 13 NIDDM and 6 non-diabetic) were characterized for immunoglobulin isotypes and light chain specificity. All ICAs were IgG-type and none were IgM- or IgA-type (median titre: 20 JDF units; range 10-160). Light chain specificity showed that 25/36 (69.4%) of the diabetic patients had lambda and kappa chains. Half of the non-diabetic subjects had both lambda and kappa chains. The rest had only lambda chains. Isotyping for ICA-IgG subclass combination with IUIS/WHO reference monoclonal antibodies in the diabetic patients gave the following: IgG1 alone-9 (25%), IgG1+2+3-8 (22.2%), IgG1+2-11 (30.6%), IgG1+3-6 (16.7%), IgG2+3-2 (5.6%). No ICA-IgG4 was detected. The frequency of the subclasses would be: IgG1-94.4%, IgG2-58.3% and IgG3-44.4%. The distribution of ICA-IgG subclasses was not affected by diabetes type (IDDM or NIDDM) or duration of disease. Of the 6 non-diabetic subjects only one had a single ICA-IgG subclass (IgG1). Serum levels of IgG subclasses in a subgroup of the patients (n = 16) were not significantly different from normal individuals. Biochemical modification of pancreatic tissue prior to ICA testing showed that acetylneuraminic acid residues, lipid and protein components were associated with binding of ICAs. The co-existence of other autoantibodies was also tested in these 42 ICA-positive sera. Twelve individuals (1 non-diabetic) had thyroid autoantibodies. Antibodies to thyrotrophin receptor, gastric parietal cell and rheumatoid factor were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

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Management of diabetic pregnancy requires objective information regarding blood glucose levels with the aim of achieving strict euglycaemia. Self-blood glucose monitoring and measurement of glycosylated haemoglobin (HbA1) are two methods widely adopted in clinical practice. Serum fructosamine assay was recently introduced as a parameter for assessing short-term glycaemic control. Our study compared serial measurement of serum fructosamine with HbA1 as indices of short-term glycaemic control in a group of diabetic pregnant women prospectively from early/mid-trimester till delivery. Patients performed self-blood glucose glucose concentration of the preceding two weeks were assessed. Our results showed that both serum fructosamine and HbA1 gave comparable information regarding short-term glycaemic control. Serum fructosamine estimation did not offer any distinct additional advantage as a retrospective index of diabetic control in the management of diabetic pregnancy.

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We previously showed that for Singapore diabetics, a low prevalence of islet cell antibodies (ICA) and insulin autoantibodies (IAA) were observed, unlike the high prevalence rates in Caucasian populations. In this report, we have measured other autoimmune markers (thyroid autoantibodies, thyrotrophin [TSH] receptor antibodies, rheumatoid factor and anti-dsDNA antibodies) to assess the extent of autoimmunity in our newly diagnosed diabetes patients and those with long-standing diabetes. Results indicate that there is a raised prevalence of thyroid autoantibodies in diabetics compared to the general population. Thyroid autoantibodies occurred in 24.3% (28/115) of the patients; thyroid microsomal antibodies (16.5%, 19/115) was much higher than for thyroglobulin antibodies (1.7%, 2/115). Prevalence rates of thyroid autoantibodies were lower in diabetics who were newly diagnosed (21.6%, 11/51), compared to those with long-standing disease (25.5%, 14/55). Three patients (3/9) with gestational diabetes were also positive for thyroid autoantibodies. TSH receptor antibodies associated with Graves' disease, were found in two patients. However, they also had thyrotoxicosis. Five patients (4.3%, 5/115) were detected with rheumatoid factor, but were clinically asymptomatic. Anti-dsDNA antibodies were not detected in any of the subjects. The presentation of thyroid autoantibodies or rheumatoid factor with age did not coincide with diabetes-associated ICA and IAA. It may mean that inspite of prevalence of subclinical autoimmunity in diabetes, autoimmunity against beta-cell lesions is not associated with the overall autoimmune tendency in our diabetes.

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