Autoantibodies to ICA12 (SOX-13) are not specific for Type I diabetes.

AIMS/HYPOTHESIS: The majority of patients with Type I (insulin-dependent) diabetes mellitus has autoantibodies to insulin, glutamic acid decarboxylase and the tyrosine phosphatase-like protein IA-2. Some patients with Type I diabetes, in particular with adult onset diabetes, have, however, only cytoplasmic islet cell antibodies that could be directed to as yet unknown beta cell autoantigens. Recently, one potential antigen, ICA12, was identified as the high mobility group (HMG) box transcription factor SOX-13. Our aim was to evaluate the diagnostic sensitivity and specificity of autoantibodies to SOX-13 for autoimmune diabetes. METHODS: Full-length SOX-13 was cloned from human brain mRNA, expressed by vitro transcription/ translation and used to detect autoantibodies by immunoprecipitation and radioligand assay in patients suffering from autoimmune diabetes or non-organ-specific autoimmune diseases. RESULTS: We found SOX-13 antibodies to be present in 11 of 125 (8.8%) patients with newly diagnosed Type I diabetes and in 3 of 43 (7.0%) patients with latent autoimmune diabetes in adults. There was no association with age, sex and a particular pattern of diabetes-specific autoantibodies. Similar frequencies of SOX-13 antibodies were found in 84 patients with rheumatic diseases (4.0-11.4%) and 211 healthy control subjects (5.2 %). CONCLUSIONS/INTERPRETATION: Our data indicate that SOX-13 represents a minor target of autoantibodies in patients with autoimmune diabetes. Because SOX-13 antibodies were found not to be disease-specific, we assume they are not helpful in improving the diagnosis and prediction of Type I diabetes.

Regulation of the diabetes-associated autoantigen IA-2 in INS-1 pancreatic beta-cells.

IA-2, a member of the protein tyrosine phosphatase family, represents a major target autoantigen in type 1 diabetes. To study the regulation of IA-2 gene expression, we used INS-1 insulinoma cells to analyze beta-cell signal transduction pathways as well as the effect of metabolic and hormonal factors involved in the regulation of the insulin secretory pathway. Quantitative competitive reverse transcriptase-polymerase chain reaction revealed that an increase of cellular cAMP mediated by forskolin (10 micromol/l, 24 h) or 3-isobutyl-1-methylxanthine (100 micromol/l, 24 h) induced maximal stimulation of IA-2 mRNA levels (451 +/- 85 and 338 +/- 86% compared with basal conditions; P < 0.001). In contrast, activation of protein kinase C (PKC) by short-term treatment with phorbol 12-myristate 13-acetate (PMA) (1 micromol/l, 6 h) did not alter IA-2 expression, whereas depletion of PKC by prolonged culturing (24 h) exerted a significant inhibition (57 +/- 24%; P < 0.05). cAMP-dependent upregulation was confirmed by the findings that glucagon (10 micromol/l, 24-48 h) increased levels of IA-2 mRNA (190 +/- 35%; P < 0.05), whereas short-term incubation with high glucose concentration showed no effect. However, prolonged incubation in high glucose (21 mmol/l) induced a time- and dose-dependent increase of IA-2 mRNA expression, reaching maximal values after 144 h (285 +/- 68%; P < 0.05). These studies demonstrate that stimuli of insulin secretion that operate by activation of adenylate cyclase generating cAMP significantly increase IA-2 gene expression. In contrast, activation of PKC by high glucose concentration or PMA exerted no effect, suggesting that IA-2 gene expression is not simply coupled to insulin secretion, but may be involved in the fine regulation of beta-cell function. These findings may be important to clarify the function of IA-2 in beta-cells and elucidate mechanisms involved in the induction of autoimmunity to IA-2.

Guided tissue regeneration with bioabsorbable barriers: intrabony defects and class II furcations.

BACKGROUND: The aim of this study was to compare the effects of guided tissue regeneration (GTR) using 2 different bioabsorbable barriers (control: polylactide acetyltributyl citrate; test: polydioxanon). The polydioxanon barrier is an experimental membrane for GTR therapy that consists of a continuous occlusive barrier that has a layer of slings on the side that is meant to face the mucoperiosteal flap. METHODS: In 21 patients with 22 pairs of similar contralateral defects (30 intrabony and 14 Class II furcation lesions), each defect was randomly assigned for treatment with either control (c) or test (t) devices. At baseline and 12 months after surgery, clinical measurements, plaque index (PI) gingival index (GI), probing depth (PD), and vertical and horizontal clinical attachment loss (CAL-V; CAL-H) and standardized radiographs were obtained. RESULTS: Barrier exposure was commonly observed in both groups. Four weeks after surgery 61% of all barriers were exposed to some extent. However, both treatments revealed a significant GI reduction (P <0.005), PD reduction (-3.08 +/- 2.29 mm [t]; -3.52 +/- 2.67 mm [c]; P <0.001) and CAL-V gain (2.44 +/- 2.29 mm [t], 2.80 mm +/- 2.21 [c]; P <0.001) 12 months after surgery in all defects. Within the intrabony defects significant bony fill (2.03 +/- 1.70 mm [t]; 1.91 +/- 1.20 mm [c]; P = 0.001), and within the furcations a significant but small CAL-H gain (0.79 +/- 0.68 mm [t]; 1.13 +/- 1.44 mm [c]; P <0.05), was observed. CONCLUSIONS: Regarding GI and PD reduction as well as CAL-V and CAL-H gain, this study failed to reveal statistically significant or clinically relevant differences between test and control 12 months postsurgically. Thus, the use of both bioabsorbable barriers in GTR therapy may be recommended.

Regenerative periodontal surgery in interproximal intrabony defects with biodegradable barriers.

AIM: The comparison of the effects of guided-tissue regeneration (GTR) using 2 different biodegradable barriers (polylactide acetyltributyl citrate; polydioxanon) in 3- and 2-wall intrabony defects. METHOD: The polydioxanon barrier is an experimental membrane for GTR therapy that consists of an continuous occlusive barrier that has a layer of slings on the side that is meant to face the mucoperiosteal flap. 15 patients provided 15 pairs of similar contralateral periodontal defects: 12 predominantly 2-wall and 18 predominantly 3-wall intrabony defects. Each defect was randomly assigned to treatment with either polylactide acetyltributyl citrate (control [c]) or polydioxanon (test [t]) devices. At baseline and 6 months after surgery, clinical measurements (P1I, GI, PPD, PAL-V) were performed. RESULTS: Barrier exposure was commonly observed in both groups (control/test): 5/4 after 7 days, 9/11 after 14 days and 11/12 after 28 days postsurgically. 4 weeks after surgery, 77% of all barriers were exposed to some extent. However, both treatments revealed a significant GI reduction (p<0.05), PPD reduction [-4.63+/-1.85 mm (t), -4.17+/-1.89 mm (c); p<0.001] and PAL-V gain [3.97+/-1.17 mm (t), 3.40 mm+/-1.40 (c); p<0.001] 6 months after surgery. Regarding GI and PPD reduction as well as PAL-V gain, there were neither statistically significant nor clinically relevant differences between test and control: similar clinical results were found 6 months after surgical treatment using both biodegradable barriers. CONCLUSIONS: Based on the results of the present study, the use of both biodegradable barriers in GTR therapy may be recommended.

Autoantibodies to adrenal cytochrome P450 antigens in isolated Addison's disease and autoimmune polyendocrine syndrome type II.

Adrenal P450 enzymes 21-hydroxylase (21OH), 17alpha-hydroxylase (17OH) and side chain cleavage enzyme (SCC) represent major target antigens in adrenal autoimmunity. To evaluate the diagnostic sensitivity of autoantibodies to recombinant adrenal antigens we established rapid and sensitive radioligand assays and compared the results with adrenocortical autoantibodies (ACA) as detected by the standard immunofluorescence test. A high prevalence of antibodies to 21OH (21OH-A) was observed in patients with isolated Addison's disease (IAD) and patients suffering from autoimmune polyendocrine syndrome type II (APS II). 21OH-A were found in 19 of 25 (76%) patients with IAD and in 34 of 40 (85%) patients with APS II. In contrast, antibodies to 17OH (17OH-A) as well as antibodies to SCC (SCC-A) were detected in 12 (30%) and 13 (33%) patients with APS II whereas only a few sera from patients with IAD had 17OH antibodies (n = 3) and SCC-A (n = 1), respectively (p < 0.0001). The majority of patients with 17OH-A (83.3%) or SCC-A (76.9%) were also found positive for 21OH-A and all three antibody specificities were positively correlated with the presence of ACA. Among 52 sera with ACA 49 (94.2%), 11 (21.2%), and 9 (17.3%) were positive for 21OH-A, 17OH-A and SCC-A, respectively. By combination of 21OH-A with 17OH-A all ACA positive individuals were identified. The availability of recombinant steroid P450 enzymes made it possible to develop radiobinding assays which allow simple, sensitive and quantitative detection of autoantibodies to defined adrenal autoantigens. We here demonstrate that autoantibodies to 21-hydroxylase are sensitive markers for autoimmune Addison's disease with and without polyglandular failure. The presence of 17OH-A or SCC-A may suggest the coexistence of or progression towards polyglandular autoimmunity.

Direct binding of thyrotropin receptor autoantibody to in vitro translated thyrotropin receptor: a comparison to radioreceptor assay and thyroid stimulating bioassay.

Graves' disease is characterized by the presence of autoantibodies to the thyrotropin receptor (TSHR), which are pathogenic and responsible for disease activity. It is well recognized that the autoantibodies are heterogeneous and recognize a number of different conformational dependent epitopes on the TSHR. In this study, we have extended our previous observations to study the interaction of Graves' disease autoantibodies with TSHR ectodomain produced by in vitro transcription and translation reaction. The specific activity of the translated TSHR ectodomain has been increased by a log fold by adding an efficient ribosome binding Kozak sequence before the translation initiation codon as well as double labelling with 35S-methionine and 35S-cysteine during the translation reaction. Addition of canine pancreatic microsomes to the translation mix showed that the glycosylation of TSHR ectodomain did not occur efficiently for the nascent receptor protein. In order to determine the specificity and sensitivity of the improved assay with nonglycosylated TSHR ectodomain, we have studied 331 sera from Graves' disease patients and as controls 100 sera from patients with nonthyroid autoimmune disorders as well as sera from 200 normal control subjects with no family history of thyroid autoimmunity. With this large cohort of sera from Graves' disease and control individuals, 25% of Graves' disease sera immunoprecipitated the dual labeled, in vitro transcribed and translated TSHR ectodomain, exceeding the 98th percentile of the control sera. There was no correlation between the autoantibodies that immunoprecipitate the in vitro translated TSHR ectodomain and those that inhibit iodinated TSH binding in the radioreceptor assay and those with biological activity in a bioassay. The data are consistent with the finding that a proportion of Graves' disease autoantibodies can interact directly with TSHR ectodomain produced by in vitro transcription and translation. However, in contrast to the wide use of similar translation and immunoprecipitation assays to measure other autoantibodies for the diagnosis of autoimmune disorders, such as type 1 diabetes, the TSHR immunoprecipitation on its own is unsuitable for diagnosis of Graves' disease.

Interexaminer reliability of the assessment of clinical furcation parameters as related to different probes.

The aim of this study was to investigate the interexaminer reliability of the assessment of clinical furcation diagnosis. Horizontal attachment level (PAL-H) measurements were obtained by 3 examiners in 6 molars in each of 10 patients with advanced periodontitis. In each patient, 3 molars were examined using a 3 mm incrementally marked Nabers probe, and 3 molars were examined using a pressure-calibrated plastic probe (TPS). Assignment of the probe was random, and the schedule of examiners was changed for each patient. Clinical assessments were validated by intrasurgical measurements in 6 patients. Sixty molars with 152 furcations were investigated. Multifactorial analysis of variance revealed that PAL-H measurements were significantly influenced by examiner and furcation location, whereas type of probe and schedule of examination had no influence. The overall intraclass correlation coefficient was r = 0.695. The difference between clinical and intrasurgical PAL-H assessment was influenced by examiner and location but not by type of probe. Approximately 70% of the total variance of PAL-H measurements was due to the variance of true values, whereas 30% of the variance may be explained by interexaminer and intraexaminer variance. The pressure-calibrated TPS probe failed to increase the interexaminer reliability of PAL-H measurements when compared to a Nabers probe.

Immunological heterogeneity in type I diabetes: presence of distinct autoantibody patterns in patients with acute onset and slowly progressive disease.

Type I diabetes mellitus may represent a heterogeneous disorder with a distinct pathogenesis in patients with young and adult onset of the disease. To investigate whether serological markers directed to different autoantigens have the potential to distinguish acute onset from slowly progressive Type I diabetes we analysed antibodies to tyrosine phosphatases IA-2/ICA512 (IA-2A) and IA-2beta/phogrin (IA2betaA), antibodies to GAD65 (GADA) and cytoplasmic islet cell antibodies (ICA) in a non-selected group of diabetic patients clinically classified as having Type I or Type II diabetes at diagnosis. Both IA-2A and IA-2betaBA were found to be positively associated with onset before the age of 20 years and the presentation of classical features of Type I diabetes. In Type I diabetes 56 % (112/200) of patients were positive for IA-2A and 38 % (76/200) for IA-2betaA. In contrast, only 1 of 785 (0.1 %) patients with Type II diabetes had IA-2A and all of them were negative for IA-2betaA (p < 0.001). Among the patients with Type II diabetes 7.6% (n = 60) were ICA positive and 2.8% (n = 22) had GADA suggesting the presence of slowly progressive Type I diabetes. GADA were found in 8 of 60 (13.3 %) ICA positive subjects which was lower than the percentage detected in patients with acute onset of diabetes (115/157 73.2%) (p < 0.001). Blocking of double antibody positive sera showed that only 3 of 8 (37.5 %) patients with slowly progressive diabetes had ICA restricted to GAD or IA-2 whereas ICA were completely inhibited in 12 of 20 (60.0 %) patients with Type I diabetes. Among 193 patients with Type II diabetes available for follow-up, 35 % of ICA positives, 58 % of GADA positives and 60 % of those positive for both markers required insulin by 3 years. However, using strict criteria for the switch to insulin treatment the corresponding sensitivity of each marker was only low (9%, 10% and 5%). We show that clinical subtypes of Type I diabetes are associated with distinct humoral autoimmunity. IA-2A and GADA were associated with classical features of Type I diabetes whereas GADA and an uncharacterized ICA subspecificity indicate slowly progressive disease.