The immunofluorescence techniques in the diagnosis of endocrine autoimmune diseases.

In the study of autoimmune diseases, the laboratory plays a very important role. We describe the immunofluorescence techniques (direct, indirect, complement-fixing, double) for determining the presence of autoantibodies and their role in the autoimmune endocrine diseases.

Measuring adrenal autoantibody response: interlaboratory concordance in the first international serum exchange for the determination of 21-hydroxylase autoantibodies.

21-hydroxylase autoantibodies (21OHAb) are the gold standard immune marker to identify patients with clinical or subclinical autoimmune Addison's disease (AAD). No assessment of interlaboratory concordance has been made for 21OHAb measurement. Serum samples from 267 patients with primary adrenal insufficiency and from 83 healthy control subjects were distributed to four independent laboratories that determined presence and titer of 21OHAb, by using radiobinding assays with either in vitro translated 35S-radiolabelled or 125I-radiolabelled autoantigen. Cohen's κ of inter-rater agreement ranged from 0.857 to 0.983, showing a very good concordance of the positive/negative score among the four laboratories. Passing-Bablok regression showed a good agreement of 21OHAb titers arranged by ranks, but important discrepancies emerged at the Bland-Altman plot, as the repeatability coefficient was much higher than the laboratory cut-offs, which indicates that results from different laboratories cannot be used interchangeably. A standardization international program for 21OHAb measurement is strongly needed.

Evaluation of the autoimmune regulator (AIRE) gene mutations in a cohort of Italian patients with autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) and in their relatives.

OBJECTIVE: Autoimmune-polyendocrinopathy-candidiasis-ectodermal-dystrophy (APECED) is a rare syndrome characterized by chronic candidiasis, chronic hypoparathyroidism and Addison's disease. APECED has been associated with mutations in autoimmune regulator (AIRE) gene. Our aim is to perform a genetic analysis of the AIRE gene in Italian APECED patients and in their relatives. Design AIRE mutations were determined by DNA sequencing in all subjects. Patients were tested for clinical autoimmune or non-autoimmune diseases, or for organ and non-organ specific autoantibodies. PATIENTS: A total of 24 Italian patients with APECED (15 from the Venetian region, 2 from Southern-Tyrol, 4 from Apulia, 3 from Sicily), 25 relatives and 116 controls were studied. RESULTS: Ten out of the 15 Venetian patients (66%) were homozygous for R257X or compound heterozygous with 1094-1106del13. One patient was homozygous for 1094-1106del13 and another for R139X. A novel mutation (1032-1033delGT) in combination with 1094-1106del13 was identified in one patient. No mutations were found in two cases. Two patients from Southern Tyrol were homozygous for R257X and for 1094-1106del13bp. All patients from Apulia were homozygous or heterozygous for W78R combined with Q358X. The patients from Sicily were homozygous for R203X or compound heterozygous with R257X. The analysis of the genotype-phenotype revealed that patients carrying 1094-1106del13 at the onset of Addison's disease were significantly older than those carrying other mutations. The genetic study of 25 relatives identified 20 heterozygous subjects. They suffered from various autoimmune and non-autoimmune diseases but no major disease of APECED was found. CONCLUSION: These data demonstrate the great genetic heterogeneity for the AIRE mutations in Italian APECED patients, and that the heterozygosity for AIRE mutations do not produce APECED.

A human monoclonal autoantibody to the thyrotropin receptor with thyroid-stimulating blocking activity.

BACKGROUND: Human monoclonal autoantibodies (MAbs) are valuable tools to study autoimmune responses. To date only one human MAb to the thyrotropin (TSH) receptor (TSHR) with stimulating activity has been available. We now describe the detailed characterization of a blocking type human MAb to the TSHR. METHODS: A single heterohybridoma cell line was isolated from the peripheral blood lymphocytes of a patient with severe hypothyroidism (TSH 278 mU/L) using standard techniques. The line stably expresses a TSHR autoantibody (5C9; IgG1/kappa). Ability of 5C9 to bind and compete with 125I-TSH or TSHR antibodies binding to the TSHR was tested using tubes coated with solubilized TSHR. Furthermore, the blocking effects of 5C9 on stimulation of cyclic AMP production was assessed using Chinese hamster ovary (CHO) cells expressing the wild-type human TSHR or TSHRs with amino acid mutations. MAIN OUTCOME: 5C9 IgG bound to the TSHR with high affinity (4 x 10(10) L/mol) and inhibited binding of TSH and a thyroid-stimulating human monoclonal autoantibody (M22) to the receptor. 5C9 IgG preparations inhibited the cyclic AMP-stimulating activities of TSH, M22, serum TSHR autoantibodies and thyroid-stimulating mouse monoclonal antibodies. Furthermore 5C9 reduced the constitutive activity of wild-type TSHR and TSHR with some activating mutations. The effect of different amino acid mutations in the TSHR on 5C9 biological activity was studied and TSHR Lys129Ala or Asp203Ala completely abolished the ability of 5C9 to block TSH-mediated stimulation of cyclic AMP production. CONCLUSIONS: The availability of 5C9 provides new opportunities to investigate the binding and biological activity of TSHR blocking type autoantibodies including studies at the molecular level. Furthermore, monoclonal antibodies such as 5C9 may well provide the basis of new drugs to control TSHR activity including applications in thyroid cancer and Graves' ophthalmopathy.

Analysis of the GAD65-GAD65 autoantibody interaction.

BACKGROUND: GAD(65)Ab are important markers of risk of development of type 1 DM. METHODS: With the need to improve the disease specificity of GAD(65)Ab measurement in mind, we have analysed the interaction between recombinant human GAD(65) and GAD(65)Ab from different groups of subjects in terms of association and dissociation rate constants and equilibrium constants. In addition, binding of GAD(65)Ab from various groups of subjects to wild-type GAD(65) versus GAD(65) containing a mutation E517P was studied. RESULTS: Affinity constants for serum GAD(65)Ab in 12 type 1 DM patients ranged from 0.9 x 10(10) L/mol to 11.2 x 10(10) L/mol and from 0.8 x 10(10) L/mol to 14.0 x 10(10) L/mol in sera from 11 individuals without type 1 DM. Serum GAD(65)Ab concentrations assessed by Scatchard analysis ranged from 0.04 to 24.8 microg/mL in type 1 DM patients (n=12) and from 0.04 to 141.8 microg/mL in individuals without type 1 DM (n=11). CONCLUSIONS: Overall, our study indicated that GAD(65)Ab in different patients studied showed similar association and dissociation rate constants and similar affinity constants. However, GAD(65)Ab concentrations vary widely between different sera. There was a modest reduction of the median binding of GAD(65)Ab to GAD(65) E517P in the group of patients with type 1 DM compared to patients without type 1 DM.

Estimated risk for developing autoimmune Addison's disease in patients with adrenal cortex autoantibodies.

CONTEXT: Patients with adrenal cortex autoantibodies (ACA) without overt autoimmune Addison's disease (AAD) are at risk of adrenal failure. DESIGN: To assess the contribution of different clinical, immunological, genetic, and functional factors in the progression to AAD, we followed up 100 ACA-positive and 63 ACA-negative patients without AAD for a maximum of 21 yr (mean 6.0 yr, median 4.8). ACA were measured by immunofluorescence and 21-OH autoantibodies (Abs) by RIA. Adrenal function was assessed by measuring basal levels of cortisol, aldosterone, ACTH, renin activity, and cortisol response to ACTH. The risk of developing AAD was calculated using survival and multivariate analyses. RESULTS: AAD developed in 31 ACA-positive patients and one ACA-negative patient. The cumulative risk of disease in ACA-positive patients was 48.5% [95% confidence interval (CI) 40.8-56.1]. The cumulative risk was higher in children than adults (100 vs. 31.9%; P < 0.0001), males than females (68.6 vs. 42.7%; P = 0.006), patients with subclinical rather than normal adrenal function at entry (87.4 vs. 30.1%; P < 0.0001), patients with hypoparathyroidism and/or candidiasis than patients with other autoimmune or nonautoimmune diseases (100 vs. 29.7%; P < 0.0001), and patients with high rather than low-medium ACA titers (62.8 vs. 41.2%; P = 0.12). The presence of human leukocyte antigen (HLA)-DRB1 did not appear to contribute to the prediction of AAD. Adjusted hazard ratios by Cox model for the development of AAD were 3.37 for males (CI 1.38-8.24), 5.23 for hypoparathyroidism and/or candidiasis (CI 1.53-17.92), 3.33 for high antibody titers (CI 1.43-7.78), and 6.15 for impaired adrenal function at entry (CI 2.79-13.57). CONCLUSIONS: These results were used to construct a risk algorithm for estimating the probability of developing AAD from the combination of gender, age, adrenal function, antibody titer, and associated autoimmune disorders at entry. The values of estimated risk could be used to decide appropriate follow-up intervals and future immunointervention strategies.

Analysis of the interaction between human steroid 21-hydroxylase and various monoclonal antibodies using comparative structural modelling.

OBJECTIVE: To study the interaction between human steroid 21-hydroxylase (21-OH) and monoclonal antibodies (MAbs) to 21-OH directed to 3 different epitopes recognised by 21-OH autoantibodies characteristic of autoimmune Addison's disease. DESIGN: Build comparative structural models of 21-OH, 21-OH MAbs and complexes of 21-OH-21-OH MAbs and study the effects of 21-OH MAbs on 21-OH enzyme activity. Then, analyse the relationship between sites important for binding of 21-OH MAbs and 21-OH autoantibodies and sites important for 21-OH enzyme activity. METHODS: Variable (V) regions of 21-OH MAbs (M21-OH1, M21-OH3, M21-OH5) were sequenced and models of the MAbs built using structures of antibodies in the database as templates. A comparative model of 21-OH was built using the crystal structure of rabbit cytochrome p450 2c5/3LVdH as template. 21-OH enzyme activity was measured in terms of conversion of [3H]progesterone to deoxycorticosterone and the effect of purified MAb IgGs on 21-OH enzyme activity was assessed. RESULTS: M21-OH1, M21-OH3 and control MAb had no effect on 21-OH enzyme activity with 88.8% +/- 24% (n = 6), 86.7% +/- 7.6% (n = 6) and 86.5% +/- 10.6% (n = 6) of activity remaining in the presence of the respective IgGs. This was consistent with the epitopes for M21-OH1 and M21-OH3 being located distant from 21-OH enzyme active sites in our 21-OH model. The epitope for M21-OH5 which inhibited 21-OH enzyme activity (48.5 +/- 8.3% activity remaining; P < 0.001 compared with control MAb IgG) was found close to the redox protein binding site in our 21-OH model. CONCLUSIONS: A comparative model of 21-OH has been produced. Analysis of experimental data in the context of the model suggests that M21-OH5 inhibits 21-OH enzyme activity through interference with redox protein binding.

Adrenal cortex autoantibodies in subjects with normal adrenal function.

The recent advances in our understanding of immunology have greatly improved our knowledge about the natural history of autoimmune diseases and, in particular, of autoimmune Addison's disease (Autoimmune AD). Autoimmune AD is a chronic disorder with a long preclinical period marked by the presence of adrenal cortex autoantibodies (ACAs). In this chapter the main data on this will be analyzed. The populations with the highest risk of Autoimmune AD are first relatives of patients with AAD and patients with autoimmune diseases, particularly those with chronic hypoparathyroidism or with premature ovarian failure. The best markers to identify the subjects at risk are ACAs detected by the immunofluorescence test on human or animal tissues, or 21-hydroxylase autoantibodies (21-OHAbs) detected by radioimmunoassay (RIA). The evaluation of adrenal cortex function in these individuals includes the basal determination of adrenocorticotropic hormone (ACTH), cortisol, aldosterone, plasma renin activity and cortisol after intravenous stimulation with synthetic ACTH. The multivariate analysis of the main factors (genetics, age, gender, titers of antibodies, pre-existing disease, status of the adrenal function) revealed that the risk of future AAD depends only on the presence of high antibody titers, chronic hypoparathyroidism or chronic candidiasis and adrenal dysfunction. On the basis of these parameters the risk of future Autoimmune AD can be calculated with an equation model. Patients with different risk scores need to be monitored at different time intervals, and those at high risk need to be strictly monitored and are the ideal subjects for future prevention trials.

Italian addison network study: update of diagnostic criteria for the etiological classification of primary adrenal insufficiency.

Primary adrenal insufficiency (PAI) is clinically evident in one in 8000 individuals. A correct etiological classification is critical for correct disease management. To update the diagnostic criteria for the etiological classification of PAI, a multicentric network was established in Italy, and 222 patients with PAI were studied. Both 21-hydroxylase and adrenal cortex autoantibodies (21OHAb and ACA, respectively) were tested in two independent laboratories on coded samples and found in 65-66% and 58-61% of cases, respectively. Autoimmune polyendocrine syndrome I was diagnosed in 11 of the 222 patients. Of the remaining 211 patients, 38 (18%) had a nonautoimmune form of PAI. In 145 subjects (65%), the presence of adrenal autoantibodies, without signs of other forms of PAI, led to a diagnosis of autoimmune Addison's disease. In six cases (3%), PAI remained idiopathic. Logistic regression analysis showed a 92.2-92.7% probability of correct reclassification for the two 21OHAb assays and 84.5-85.9% for the ACA assays. We conclude that the simultaneous presence of both 21OHAb and ACA permits unambiguous diagnosis of autoimmune Addison's, whereas subjects with low antibody titers should undergo both instrumental and biochemical tests to exclude other causes of PAI. Lastly, we developed a comprehensive flowchart for the classification of PAI for use in routine clinical practice.

Autoantibodies to human tryptophan hydroxylase and aromatic L-amino acid decarboxylase.

OBJECTIVE: To assess the prevalence of autoantibodies (Abs) to tryptophan hydroxylase (TPH) and aromatic l-amino acid decarboxylase (AADC) in patients with different autoimmune diseases and to analyse their respective epitopes. DESIGN: TPH and AADC Abs were measured in an immunoprecipitation assay using (35)S-labelled full-length and fragments of TPH and AADC. METHODS: Patients with different autoimmune adrenal diseases (n=84), non-adrenal autoimmune diseases (n=37), idiopathic vitiligo (n=8) and 56 healthy blood donors were studied. RESULTS: Fourteen of twenty-three (61%) of patients with autoimmune polyglandular syndrome (APS) type I and 1/34 (3%) of patients with isolated Addison's disease (AD) were positive for TPH Abs. None of the patients with APS type II (n=27), coeliac disease (n=10), autoimmune thyroid disease (AITD) (n=11), type 1 diabetes mellitus (DM) (n=16) or idiopathic vitiligo (n=8) was positive for TPH Abs. AADC Abs were detected in 12/23 (52%) patients with APS type I, in 1/29 (3%) patients with APS type II and 1/34 (3%) patients with isolated AD. None of the patients with coeliac disease, type 1 DM, AITD or idiopathic vitiligo was positive for AADC Abs. TPH Abs were found to interact with the C-terminal amino acids (aa) 308-423, central aa 164-205 and N-terminal aa 1-105 of the TPH molecule. AADC Ab binding epitopes were within the C-terminal aa 382-483, the central aa 243-381 and the N-terminal aa 1-167. CONCLUSIONS: Our study suggests that TPH Abs and AADC Abs react with several different epitopes and that different epitopes are recognized by different sera. The prevalence of TPH Abs and AADC Abs in patients with APS type I in our study is in agreement with previous reports. TPH Abs and AADC Abs were found very rarely in patients with other forms of autoimmune adrenal disease and were not detected in patients with non-adrenal autoimmune diseases.

Update on autoimmune polyendocrine syndromes (APS).

Autoimmune Polyendocrine Syndromes (APS) were initially defined as a multiple endocrine gland insufficiency associated to an autoimmune disease in a patient. Neufeld & Blizzard (1980) suggested a classification of APS, based on clinical criteria only, describing four main types. APS-1 is characterized by presence of chronic candidiasis, chronic hypoparathyroidism, Addison's disease. It is a very rare syndrome interesting young subjects correlating to different mutations of AIRE (AutoImmuneRegulator) gene on chromosome 21. APS-2 is characterized by presence of Addison's disease (always present), autoimmune thyroid diseases and/or type 1 diabetes mellitus. It is a rare syndrome interesting particularly adult females and associated to a genetic pattern of HLA DR3/DR4. Autoimmune thyroid diseases associated to other autoimmune diseases (excluding Addison's disease and/or hypoparathyroidism), are the main characteristics of APS-3. The different clinical combinations of autoimmune diseases not included in the previous groups are characteristics of APS-4. In this paper criteria for defining a disease as autoimmune are presented. Furthermore, the classification, epidemiology, pathogenesis, genetic, animal models, clinical features, laboratory's tests, imaging, therapy, recent progresses in understanding the APS and a detailed analysis of large group of our patients affected by different types of APS are proposed and discussed.

A sensitive non-isotopic assay for GAD65 autoantibodies.

BACKGROUND: A new ELISA for 65 kDa isoform of glutamic acid decarboxylase autoantibodies (GAD(65) Abs), which depends on GAD(65) Ab acting divalently and forming a bridge between immobilized GAD(65) and liquid-phase GAD(65)-biotin, is described. METHODS: Sera (25 microl) were incubated in GAD(65)-coated ELISA plate wells followed by washing and incubation with GAD(65)-biotin. After a further wash step, GAD(65)-biotin bound was quantitated by addition of streptavidin peroxidase followed by tetramethylbenzidine. Assay calibration was with WHO reference preparation 97/550. RESULTS: Using a cut-off for positivity of 5 units/ml, sera from 0.7% (2/300) healthy blood donors (HBDs), 100% (39/39) selected type 1 diabetes mellitus (DM) patients, 1.6% (1/62) type 2 diabetes mellitus patients and 3% (4/119) autoimmune disease controls were GAD(65) Ab positive in the ELISA. Levels of positivity in an immunoprecipitation assay (IPA) based on 125I-labelled GAD(65) (cut-off 25 units/ml) were 1%, 82%, 0% and 3%, respectively. ELISA inter-assay coefficients of variation (n=12) were 7.3%, 3.8%, 7.2% and 6.3% at 5.4, 40.8, 137 and 382 units/ml, respectively. CONCLUSIONS: The ELISA we have described has sensitivity and specificity at least as high as current radioactive assays. It has good precision and handling making it suitable for routine use.

Autoimmune adrenal insufficiency and autoimmune polyendocrine syndromes: autoantibodies, autoantigens, and their applicability in diagnosis and disease prediction.

Recent progress in the understanding of autoimmune adrenal disease, including a detailed analysis of a group of patients with Addison's disease (AD), has been reviewed. Criteria for defining an autoimmune disease and the main features of autoimmune AD (history, prevalence, etiology, histopathology, clinical and laboratory findings, cell-mediated andhumoral immunity, autoantigens and their autoepitopes, genetics, animal models, associated autoimmune diseases, pathogenesis, natural history, therapy) have been described. Furthermore, the autoimmune polyglandular syndromes (APS) associated with AD (revised classification, animal models, genetics, natural history) have been discussed. Of Italian patients with primary AD (n = 317), 83% had autoimmune AD. At the onset, all patients with autoimmune AD (100%) had detectable adrenal cortex and/or steroid 21-hydroxylase autoantibodies. In the course of natural history of autoimmune AD, the presence of adrenal cortex and/or steroid 21-hydroxylase autoantibodies identified patients at risk to develop AD. Different risks of progression to clinical AD were found in children and adults, and three stages of subclinical hypoadrenalism have been defined. Normal or atrophic adrenal glands have been demonstrated by imaging in patients with clinical or subclinical AD. Autoimmune AD presented in four forms: as APS type 1 (13% of the patients), APS type 2 (41%), APS type 4 (5%), and isolated AD (41%). There were differences in genetics, age at onset, prevalence of adrenal cortex/21-hydroxylase autoantibodies, and associated autoimmune diseases in these groups. "Incomplete" forms of APS have been identified demonstrating that APS are more prevalent than previously reported. A varied prevalence of hypergonadotropic hypogonadism in patients with AD and value of steroid-producing cells autoantibodies reactive with steroid 17alpha-hydroxylase or P450 side-chain cleavage enzyme as markers of this disease has been discussed. In addition, the prevalence, characteristic autoantigens, and autoantibodies of minor autoimmune diseases associated with AD have been described. Imaging of adrenal glands, genetic tests, and biochemical analysis have been shown to contribute to early and correct diagnosis of primary non-autoimmune AD in the cases of hypoadrenalism with undetectable adrenal autoantibodies. An original flow chart for the diagnosis of AD has been proposed.