Diagnostic Value of Autoantibodies against Steroidogenic Enzymes and Hormones in Infertile Women with Premature Ovarian Insufficiency.

The objective of the study was to evaluate the profile and diagnostic significance of serum autoantibodies in infertile patients with premature ovarian insufficiency (POI). The pilot study included 26 patients of reproductive age with POI and diminished ovarian reserve who received complex treatment using new surgical technologies (Group 1) and 18 patients without POI (Group 2). The profile of serum autoantibodies, including anti-ovarian antibodies, antibodies against thyroid peroxidase (TPO), steroidogenic enzymes, and steroid and gonadotropic hormones, was studied using modified ELISAs and human recombinant steroidogenic enzymes (CYP11A1, CYP19A1, CYP21A2). Patients in Group 1 had higher levels of IgG autoantibodies against steroidogenic enzymes, estradiol, progesterone, and TPO than those in Group 2. Tests for IgG antibodies against CYP11A1, CYP19A1, and CYP21A2 exhibited high sensitivity (65.4-76.9%), specificity (83.3-89.9%), and AUC values (0.842-0.910) for POI, the highest in the first test. Three-antibodies panel screening showed higher diagnostic accuracy (84.1% versus 75-79.6%). The levels of these antibodies correlated with menstrual irregularities and a decrease in the antral follicle count. Thus, antibodies against CYP11A1, CYP19A1, and CYP21A2 have a high diagnostic value for POI. Three-antibody panel screening may improve the accuracy of POI diagnosis and be useful for identifying high-risk groups, early stages of the disease, and predicting POI progression.

Reversible primary adrenal insufficiency related to anti-programmed cell-death 1 protein active immunotherapy: Insight into an unforeseen outcome of a rare immune-related adverse event.

The immune checkpoint inhibitors (ICPi) revolutionize the cancer therapeutics, though not being devoid of toxicity. The immune-related primary adrenal insufficiency (PAI) is a rare, yet potentially life-threatening, adverse event, posing diagnostic and therapeutic challenges. We report the first case of reversible PAI related to nivolumab (programmed cell-death 1 protein inhibitor) in a 42-year-old male with metastatic rectal adenocarcinoma. PAI manifested as profound fatigue, disorientation, hypotension, hyperpigmentation of palmar creases, and hyponatremia without hyperkalemia 16 weeks after initiation of nivolumab. Due to impending adrenal crisis, intravenous stress doses of hydrocortisone and hydration with normal saline were initiated. When the state of patient was stabilized, PAI was confirmed through 250 µg Synacthen test 24 h after temporary cessation of hydrocortisone. Hydrocortisone was fixed at maintenance dose, while mineralocorticoid substitution was not required. PAI was ascribed to nivolumab based on history, physical examination, and laboratory work-up with emphasis on positivity of anti-21-hydroxylase antibodies and exclusion of other causes of PAI by normal imaging of adrenal glands on computed tomography (CT). Reevaluation of adrenal function during follow up demonstrated complete recovery. A review of literature concerning the immune-related PAI indicated that the complete recovery of adrenal function, the normal CT imaging, and the positivity of anti-21-hydroxylase antibodies observed in our patient are exceptional findings of immune-related PAI. Finally, heightened suspicion of immune-related PAI in case of hyponatremia without hyperkalemia and constant vigilance for diagnosis of rare, but real, reversibility of immune-related PAI are of paramount importance.

Fatigue in Primary Sjögren's Syndrome: Clinical, Laboratory, Psychometric, and Biologic Associations.

OBJECTIVE: To identify independent contributors of fatigue in primary Sjögren's syndrome (SS) patients, taking into account clinical, laboratory, and psychological features, and to explore the potential role of interferon (IFN)-induced gene indoleamine 2,3-dioxygenase (IDO-1), anti-21-hydroxylase (anti-21[OH]) antibodies, and soluble BAFF. METHODS: Detailed clinical and laboratory characteristics were recorded for 106 primary SS patients. The Functional Assessment of Chronic Illness Therapy-Fatigue, Zung Depression Scale, State-Trait Anxiety Inventory, Eysenck Personality Questionnaire Scale, and Athens Insomnia Scale were adopted to assess fatigue, depression, anxiety, and sleep disturbances, respectively. Peripheral whole blood expression levels of IDO-1, as well as type I and II IFN-induced genes were calculated using quantitative reverse transcriptase-polymerase chain reaction. Serum anti-21(OH) antibodies and soluble BAFF levels were determined by a radioimmunoassay and an enzyme-linked immunosorbent assay, respectively. Univariate and multivariate models were performed to identify determinants of fatigue. RESULTS: Fatigue was detected in 32 of 106 (30.2%) primary SS patients. In univariate analysis, fatigue was associated with arthralgias/myalgias, fibromyalgia hydroxychloroquine therapy, both state and trait anxiety scores, depression, and neuroticism, as well as impaired sleep patterns. Multivariate analysis revealed neuroticism (odds ratio [OR] 6.9, [95% confidence interval (95% CI) 1.7-28.0]), depression (OR 3.0 [95% CI 0.8-11.0]), and fibromyalgia (OR 5.5 [95% CI 1.1-27.7]) as independent fatigue contributors. Soluble BAFF levels, anti-21(OH) autoantibodies, and IDO-1 messenger RNA expression did not significantly differ between fatigued and nonfatigued primary SS patients. CONCLUSION: Depression, neuroticism, and fibromyalgia play a major role in primary SS-associated fatigue and should be addressed in clinical practice, with active collaboration between rheumatologists and mental health professionals. Further studies are warranted in order to explore underlying pathophysiologic pathways that might explain fatigue in the setting of primary SS.

Design and Measurement of Nonislet-Specific Autoantibodies for the Type 1 Diabetes Genetics Consortium Autoantibody Workshop.

The Type 1 Diabetes Genetics Consortium (T1DGC) comprised groups of investigators from many countries throughout the world, with a common goal of identifying genes predisposing to type 1 diabetes. The T1DGC ascertained and collected samples from families with two or more affected siblings with type 1 diabetes and generated a broad array of clinical, genetic, and immunologic data. The T1DGC Autoantibody Workshop was designed to distribute data for analyses to discover genes associated with autoantibodies in those with type 1 diabetes. In the T1DGC-affected sibling pair families, three T1DGC Network laboratories measured antibodies to the islet autoantigens GAD65 and the intracellular portion of protein tyrosine phosphatase (IA-2A). The availability of extensive genetic data provided an opportunity to investigate the associations between type 1 diabetes and other autoimmune diseases for which autoantibodies could be measured. Measurements of additional nonislet autoantibodies, including thyroid peroxidase, tissue transglutaminase, 21-hydroxylase, and the potassium/hydrogen ion transporter H+/K+-ATPase, were performed by the T1DGC laboratory at the Barbara Davis Center for Childhood Diabetes, Aurora, CO. Measurements of all autoantibodies were transmitted to the T1DGC Coordinating Center, and the data were made available to members of the T1DGC Autoantibody Working Groups for analysis in conjunction with existing T1DGC genetic data. This article describes the design of the T1DGC Autoantibody Workshop and the quality-control procedures to maintain and monitor the performance of each laboratory and provides the quality-control results for the nonislet autoantibody measurements.

High frequency of cytolytic 21-hydroxylase-specific CD8+ T cells in autoimmune Addison's disease patients.

The mechanisms behind destruction of the adrenal glands in autoimmune Addison's disease remain unclear. Autoantibodies against steroid 21-hydroxylase, an intracellular key enzyme of the adrenal cortex, are found in >90% of patients, but these autoantibodies are not thought to mediate the disease. In this article, we demonstrate highly frequent 21-hydroxylase-specific T cells detectable in 20 patients with Addison's disease. Using overlapping 18-aa peptides spanning the full length of 21-hydroxylase, we identified immunodominant CD8(+) and CD4(+) T cell responses in a large proportion of Addison's patients both ex vivo and after in vitro culture of PBLs ≤20 y after diagnosis. In a large proportion of patients, CD8(+) and CD4(+) 21-hydroxylase-specific T cells were very abundant and detectable in ex vivo assays. HLA class I tetramer-guided isolation of 21-hydroxylase-specific CD8(+) T cells showed their ability to lyse 21-hydroxylase-positive target cells, consistent with a potential mechanism for disease pathogenesis. These data indicate that strong CTL responses to 21-hydroxylase often occur in vivo, and that reactive CTLs have substantial proliferative and cytolytic potential. These results have implications for earlier diagnosis of adrenal failure and ultimately a potential target for therapeutic intervention and induction of immunity against adrenal cortex cancer.

Screening for X-linked adrenoleukodystrophy among adult men with Addison's disease.

OBJECTIVES: X-linked adrenoleukodystrophy is an important cause of Addison's disease in boys, but less is known about its contribution to Addison's disease in adult men. After surveying all known cases of X-linked adrenoleukodystrophy in Norway in a separate study, we aimed to look for any missed cases among the population of adult men with nonautoimmune Addison's disease. STUDY DESIGN: Among 153 adult men identified in a National Registry for Addison's Disease (75% of identified male cases of Addison's disease in Norway), those with negative indices for 21-hydroxylase autoantibodies were selected. Additionally, cases with low autoantibody indices (48-200) were selected. Sera from subjects included were analysed for levels of very long-chain fatty acids, which are diagnostic for X-linked adrenoleukodystrophy in men. RESULTS: Eighteen subjects had negative indices and 17 had low indices for 21-hydroxylase autoantibodies. None of those with low indices and only one of those with negative indices were found to have X-linked adrenoleukodystrophy; this subject had already been diagnosed because of the neurological symptoms. Cases of Addison's disease proved to be caused by X-linked adrenoleukodystrophy constitute 1·5% of all adult male cases in Norway; the proportion among nonautoimmune cases was 15%. CONCLUSIONS: We found X-linked adrenoleukodystrophy to be an uncommon cause of Addison's disease in adult men. However, this aetiological diagnosis has far-reaching consequences both for the patient and for his extended family. We therefore recommend that all adult men with nonautoimmune Addison's disease be analysed for levels of very long-chain fatty acids.

[Subclinical adrenal diseases: silent pheochromocytoma and subclinical Addison's disease]. / Maladies surrénaliennes infracliniques: cas du phéochromocytome silencieux et de la maladie d'Addison infra-clinique.

The silent pheochromocytoma, a hidden form of pheochromocytoma, exposes the patient to an increased risk of mortality if the diagnosis is not established on time. Biological diagnosis of pheochromocytoma can be difficult. Catecholamine secretion is dependent on tumor size and a large number of physiological, pharmacological, lifestyle modifications and sampling conditions influence the measurement of urinary and plasma metanephrines. The prevalence of pheochromocytoma is 2% among adrenal incidentaloma smaller than 3 cm (2/3 of tumors). Recent studies suggest the almost zero risk of pheochromocytoma among these tumors if they are hypodense (<10 housefield units) on adrenal tomography. Addison's disease is a pathology affecting about 1 in 8000. Immunopathology is still unknown, but some elements advocated the hypothesis of a predominant cell-mediated immunity in particular Interferon-gamma production by CD4 T lymphocytes in the presence of an epitope from the 21-hydroxylase, as well as IgG1 subtype produced by activated B lymphocytes, autoantibodies do appear to be a simple marker of the disease. Subclinical Addison's disease is defined by the presence of anti-21-hydroxylase autoantibodies, without clinical symptoms. It evolves faster to the clinical phase in young subjects, male, having high levels of autoantibodies and with an initially impaired adrenal function. Dosage of ACTH, plasma renin active, and basal cortisol and after Synacthen allow to discriminate the subjects with low or high risk of evolution and establish an appropriate monitoring.

Brief report: adrenal autoimmunity in primary Sjögren's syndrome.

OBJECTIVE: To evaluate the prevalence of antibodies to 21-hydroxylase (anti-21[OH]), a marker of autoimmune adrenal disease, in a cohort of patients with primary Sjögren's syndrome (SS) and to investigate whether the presence of anti-21(OH) correlates with clinical, serologic, and salivary gland features of the disease. METHODS: Sera from 63 consecutive patients with primary SS, 32 patients with autoimmune thyroid disease (AITD), and 20 healthy controls were obtained and anti-21(OH) levels were determined by radioimmunoassay. Clinical, serologic, and histopathologic features were recorded, and a short Synacthen test was used to assess adrenal function reserve. Seven available minor salivary gland (MSG) tissue specimens from patients in the primary SS cohort were also assessed for interferon-α (IFNα), BAFF, and interleukin-21 (IL-21) cytokine transcripts, which are all implicated in B cell activation. RESULTS: Anti-21(OH) positivity was detected in 17.5% and 28.1% of primary SS and AITD patients, respectively, and in none of the healthy controls. While no evidence of adrenal insufficiency was detected in any of the patients studied, a blunted rate of increase in cortisol levels was observed in patients with detectable serum autoantibodies against 21(OH), compared to their anti-21(OH)-negative counterparts. A strong correlation between the serum titer of anti-21(OH) antibodies and expression of IFNα, BAFF, and IL-21 messenger RNA in MSG tissues was also detected. CONCLUSION: Adrenal autoimmunity occurs in almost 20% of patients with primary SS in association with markers of B cell activation. Although the presence of adrenal autoantibodies was not associated with adrenal insufficiency in the present study, there was a blunted adrenal response, suggesting the need for further followup and monitoring of adrenal function in patients with primary SS who are positive for the autoantibodies.

Endocrine alterations in primary Sjogren's syndrome: an overview.

Involvement of several components of the endocrine system has been proposed as significant player in primary Sjogren's syndrome (SS) pathogenesis and clinical expression. Hypoactivity of the hypothalamic-pituitary-adrenal (HPA) axis has been previously demonstrated in patients with primary SS as a result either of a pituitary defect and/or of adrenal gland dysfunction. In support of the latter hypothesis, antibodies to 21-hydroxylase (OH)--a marker of autoimmune adrenal disease--have been detected in sera from approximately one fifth of primary SS patients, in association with B-cell activating cytokines and adrenal hyporesponsiveness. As a result of HPA hypofunction, adrenal androgens and particularly dehydroepiandrosterone-sulfate (DHEA-S) have been reportedly low in primary SS individuals. Epithelial salivary gland cells undergo apoptosis in lack of both estrogens and active androgens. In the absence of a compensatory action of the latter, menopausal status can lead to salivary gland apoptotic process triggering an aberrant immune response. On the other hand, given that salivary gland tissue remodeling has been shown to be under androgenic control, the observed androgen deficiency in these patients might account for the observed alterations in the salivary gland architecture. Heightened serum and salivary gland tissue prolactin levels in primary SS patients have been also suggested as contributors in disease pathogenesis. Finally, autoimmune thyroid disease (ATD) occurs quiet commonly in the setting of primary SS and subclinical hypothyroidism is the main functional abnormality observed in these patients.

Prevalence of parietal cell antibodies in a large cohort of patients with autoimmune thyroiditis.

BACKGROUND: Autoimmune thyroiditis (AIT) may be associated with other organ-specific autoimmune disorders, including autoimmune gastritis, but the prevalence of this association is not entirely quantified. The aim of this study was to investigate the prevalence of parietal cell antibodies (PCA) in a large cohort of consecutive patients with AIT. METHODS: We retrospectively studied 2016 consecutive women and 258 men with AIT seen at our referral center in the period from 2004 to 2008. All patients were screened for the presence of PCA in the serum. RESULTS: The prevalence of serum PCA in female patients was 29.7% and progressively increased from 13% in the first-second decade of life to peak at 42% in the ninth decade. During follow up, 21.1% of the PCA-positive patients converted to PCA-negative status. Mean (±standard deviation) basal PCA levels in this group were significantly lower (32 ± 28 U/mL) compared with those remaining PCA positive (129 ± 200 U/mL). A similar prevalence (29.8%) with a similar age-dependency was found in male patients. CONCLUSIONS: In conclusion, our study demonstrates a high, age-dependent prevalence of PCA in an unselected large population of patients with AIT.

[Prevalence of anti-thyroid peroxidase and anti-adrenal 21-hidroxylase in type 1 diabetes patients]. / Prevalência do anti-TPO e anti-21-hidroxilase no paciente diabético tipo 1.

UNLABELLED: There is still no consensus about the best strategy to screen Addison's disease (AD) in type 1 diabetes mellitus (T1DM) patients. OBJECTIVE: The aim of this study was to determine the frequency of anti-21-hydroxilase (anti-21OH) in a multiethnic T1DM population and investigate if its presence is associated with any adrenal dysfunction or thyroid autoimmunity. METHODS: Forty individuals underwent an interview and blood was drawn for anti-thyroperoxidase (anti-TPO), anti-21OH, TSH, free T4 and cortisol measurement. RESULTS: Anti-21OH was found in 7.5% (n = 3), none with adrenal dysfunction. This antibody was not exclusively seen in patients with anti-TPO (+). Anti-TPO was positive in 25% and associated with higher TSH levels (p = 0.034) and older age (p = 0.009). CONCLUSIONS: Although the frequency of anti-TPO in this sample was similar to previous studies, a higher prevalence of anti-21-OH was found. However, no coexisting adrenal dysfunction was detected, which does not support universal screening for AD in this group.

The purification and application of biologically active recombinant steroid cytochrome P450 21-hydroxylase: the major autoantigen in autoimmune Addison's disease.

In primary adrenocortical failure (Addison's disease) caused by autoimmunity, autoantibodies to the steroidogenic cytochrome P450 enzyme 21-hydroxylase (21OH) are detected in the majority of patients. It is currently uncertain whether the autoantibodies themselves participate in the pathogenesis, or if they merely reflect an on-going T cell mediated response. The identification of T cells reactive with 21OH, if any, has been hampered by the lack of a high-quality antigen. In the current study recombinant human 21OH has been expressed in Spodoptera frugiperda insect cells using a baculovirus expression system. Recombinant enzymatically active 21OH was purified to apparent homogeneity by immobilized metal ion affinity chromatography. The purified enzyme was highly immunogenic in immunized SJL/J mice, and immune responses to 21OH-derived peptides assayed as T cell proliferation and interferon gamma production could be invoked after priming with the recombinant protein. Furthermore, purified 21OH was recognized by sera from patients with autoimmune Addison's disease, and it could block the binding of radiolabeled in vitro translated 21OH in a sensitive fluid-phase radioimmunoassay. We conclude that the recombinant preparation of 21OH presented here is of sufficient purity and quality to be used for studies of cellular and humoral immunity in autoimmune Addison's disease.

HLA antigen expression in autoimmune endocrinopathies.

The HLA allelic frequency was determined in three groups of autoimmune endocrinopathies: A) 30 patients with autoimmune thyroiditis, B) 20 patients with polyglandular activation of autoimmunity, and C) 10 patients with the autoimmune polyglandular syndrome type II. The groups were defined by the clinical state and serological parameters. Healthy blood donors of Caucasian population from the US database of HLA frequencies served as the controls. In group A, a higher occurrence of HLA-A24 (21.7 %) was found as compared to group B (5.0 %) and to the controls (8.5 %), of HLA-B27 (15.0 %) and of HLA-DR-11 (20 %) as compared to the controls (4.2 % and 8.5 %). In group B, a higher occurrence of HLA-A3 (25.0 %) was found as compared to group A (10 %) and to the controls (11.8 %), and of HLA-B8 (22.5 %) as compared to group A (8.3 %) and to the controls (8.6 %). In this group the occurrence of HLA-DR3 (30.0 %) was higher as compared to group A (10.0 %) and to the controls (9.8 %) and of HLA-B8 (30.0 %) as compared to group A (8.3 %) and to the controls (8.6 %). Genetic markers indicate a similarity of groups B and C. Patients in these groups could be at different stages of the same disease, however, some distinctions between them lead us to consider the possibility whether different epigenetic factors could extend the difference between these groups in the course of clinical development.

Prevalence of adrenal antibodies in Addison's disease among north Indian Caucasians.

OBJECTIVE: The prevalence of antibodies in different organ-specific autoimmune diseases can vary depending on the racial group studied. Data on the prevalence of antibodies against steroidogenic enzymes in Addison's disease is available only for white Caucasians. We have evaluated the frequency of antibodies against adrenal cytoplasm, 21-hydroxylase, 17-alpha-hydroxylase and side-chain cleavage enzyme in a cohort of Indian patients with Addison's disease of idiopathic and granulomatous aetiology. DESIGN: Study of all patients with Addison's disease on whom serum samples were available (84% of total), presenting to the Endocrinology Department in a teaching hospital in India, between 1990 and 1999. PATIENTS: Thirty-eight patients with Addison's disease (19 idiopathic, 19 granulomatous). METHODS: A radiobinding assay using in vitro transcribed and translated recombinant human 35S-labelled 21-hydroxylase, 17-alpha-hydroxylase and side-chain cleavage enzymes was utilized to detect the respective antibodies. Adrenal cytoplasmic antibodies were measured by indirect immunofluorescence on cryostatic sections of human adrenal cortex. RESULTS: Of the 19 patients with idiopathic Addison's disease, adrenal cytoplasmic antibodies were present in five (26%) patients, while 21-hydroxylase antibodies were present in four (21%) subjects. The frequency of 21-hydroxylase antibodies was similar among patients with isolated idiopathic Addison's disease (3/13, 23%), and those associated with other organ-specific autoimmune diseases (1/6, 17%). 17-alpha-hydroxylase and side-chain cleavage antibodies were present in four (21%) and three (16%) patients, respectively. Overall, at least one of the three antibodies was present in eight (42%) subjects. All four female patients with premature ovarian failure had antibodies against 17-alpha-hydroxylase and/or side-chain cleavage enzyme. Two (11%) patients with granulomatous Addison's disease had adrenal antibodies. Of these, one patient with enlarged and calcified adrenal gland secondary to tuberculosis had a high titre of antibodies against all three steroidogenic enzymes. CONCLUSIONS: Antibodies to 21-hydroxylase enzyme are less frequent in idiopathic Addison's disease in north Indians, when compared with other Caucasians. In contrast, the prevalence of 17-alpha-hydroxylase and side-chain cleavage enzyme antibodies is similar to those reported. High titre antibodies against steroidogenic enzymes may occasionally be present in patients with clinical evidence of tuberculous Addison's disease.

Autoantibodies against recombinant human steroidogenic enzymes 21-hydroxylase, side-chain cleavage and 17alpha-hydroxylase in Addison's disease and autoimmune polyendocrine syndrome type III.

OBJECTIVE: To evaluate the frequency of autoantibodies (Ab) against 21 hydroxylase (21OH), side-chain cleavage (SCC) and 17alpha-hydroxylase (17OH), in Addison's disease (AD) and autoimmune polyendocrine syndrome type III (APSIII). DESIGN AND METHODS: We used radiobinding assays and in vitro translated recombinant human (35)S-21OH, (35)S-SCC or (35)S-17OH and studied serum samples from 29 AD (18 idiopathic, 11 granulomatous) and 18 APSIII (autoimmune thyroid disease plus type 1 diabetes mellitus, without AD) patients. Results were compared with those of adrenocortical autoantibodies obtained with indirect immunofluorescence (ACA-IIF). RESULTS: ACA-IIF were detected in 15/18 (83%) idiopathic and in 1/11 (9%) granulomatous AD subjects. 21OHAb were found in 14/18 (78%) idiopathic and in the same (9%) granulomatous AD subject. A significant positive correlation was shown between ACA-IIF and 21OHAb levels (r(2)=0.56, P<0.02). The concordance rate between the two assays was 83% (24/29) in AD patients. SCCAb were found in 5/18 (28%) idiopathic (4 of whom were also positive for 21OHAb) and in the same (9%) granulomatous AD subject. 17OHAb were found in only 2/18 (11%) idiopathic and none of the granulomatous AD patients. Two APSIII patients were positive for ACA-IIF, but only one was positive for 21OHAb and SCCAb. 17OHAb were found in another two APSIII patients. CONCLUSIONS: Measurement of 21OHAb should be the first step in immune assessment of patients with AD and individuals at risk for adrenal autoimmunity, in addition to ACA-IIF. Due to their low prevalence in AD, measurement of SCCAb and 17OHAb should be indicated only for 21OHAb negative patients and/or for those with premature ovarian failure, regardless of ACA-IIF results.

Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51.

The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not. When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity. Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K. Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6. Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18. The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19. However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation. Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards. Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected.

Autoantibody binding to steroid 21-hydroxylase--effect of five mutations.

Steroid 21-hydroxylase (21-OH) is a key haem containing steroidogenic enzyme and a major adrenal specific autoantigen. Cys 428 in 21-OH is thought to have an important role in haem binding and we now describe the effects of mutations at Cys 428 (to Ser, Arg and Phe) on 21-OH autoantibody binding. Expression of wild type and mutated 21-OH was carried out using an in vitro transcription/translation (TnT) system and reactivity of 21-OH autoantibodies with mutated 21-OH analysed by western blotting (in the case of unlabelled proteins) or immunoprecipitation assay (IPA) (in the case of 35S-labelled proteins). All 3 substitutions at Cys 428 had similar effects on 21-OH autoantibody binding and each one caused a reduction in autoantibody binding to about 50% of wild type in the case of IPA and to about 70% of wild type in the case of western blotting analysis. In addition to mutations at Cys 428, we studied 2 naturally occurring mutations at Pro 30 to Leu and Ile 172 to Asn which are associated with diminished 21-OH enzyme activity. The Pro 30 mutation had no effect, but the Ile 172 mutation caused a reduction in 21-OH autoantibody binding in the IPA to about 80% of wild type. Overall, our studies emphasise the close relationship between the 21-OH aminoacid sequences important for 21-OH enzyme activity and 21-OH autoantibody binding.

The HLA-A3, Cw6,B47,DR7 extended haplotypes in salt losing 21-hydroxylase deficiency and in the Old Order Amish: identical class I antigens and class II alleles with at least two crossover sites in the class III region.

The HLA-B47,DR7 haplotype in congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency contains a deletion of most of the active CYP21 gene and the entire adjacent C4B gene. The C4A gene produces a protein which is electrophoretically C4A but antigenically C4B. In the Old Order Amish, the HLA-B47,DR7 haplotype contains no deletion, but is immunologically identical to the CAH haplotype in both areas flanking the crossover region. We compared some of the genes in the MHC Class II and Class III regions in the Amish and CAH-linked haplotypes to define further the relationships between the two. The complement factor B (Bf) proteins differed, but no Bf RFLPs were identified. The complement factor 2 genes exhibited different BamHI RFLPs. Analyses of the tumor necrosis factor-alpha genes revealed the same NcoI restriction patterns. The RD genes contained microsatellites of the same size. Portions of the MHC Class II DR and DQ, and Class III CYP21 and C4 alleles were sequenced. The exon 2 sequences of DQ2 and DR7 were identical in the two haplotypes. In the Amish haplotype, both CYP21 and C4 gene pairs were present and functionally normal. The CAH haplotype had two sequence crossovers: from CYP21P to CYP21 in the 7th intron, and from C4A to C4B between codons 1106 (exon 26) and 1157 (exon 28). A model is proposed which accounts for the CAH-linked mutant haplotype arising from a nonmutant homologue via three crossings-over.

Immunoprecipitation assay for autoantibodies to steroid 21-hydroxylase in autoimmune adrenal diseases.

Adrenal autoantibodies characteristic of autoimmune Addison disease are directed towards steroid 21-hydroxylase (21-OH; EC 1.14.99.10). We describe a new assay to measure 21-OH autoantibodies (21-OH Abs), based on immunoprecipitation by the antibodies of 35S-labeled human 21-OH. Using this immunoprecipitation assay (IPA), we detected 21-OH Abs in 42 of 64 (66%) patients with Addison disease and in 14 of 19 (74%) patients with autoimmune polyendocrine syndromes type I and type II. No 21-OH Abs were detected by the IPA in any patients with Addison disease attributable to tuberculosis (n = 9) or adrenoleukodystrophy (n = 9) or in patients with autoimmune thyroid disease (n = 28), systemic lupus erythematosus (n = 10), myasthenia gravis (n = 10), rheumatoid arthritis (n = 10), or insulin-dependent diabetes mellitus (n = 12). None of the 26 sera from healthy normal blood donors was positive for 21-OH Abs by the assay. We found good agreement between 21-OH Abs measured by IPA and by Western blotting (r = 0.83, n = 123, P < 0.001). The inter- and intraassay CVs for IPA were well < 10% at high, medium, and low concentrations of 21-OH Abs. Overall, our studies indicate that the IPA provides a specific, sensitive, and convenient system for measuring 21-OH Abs.

21-Hydroxylase, a major autoantigen in idiopathic Addison's disease.

Sera from patients with idiopathic Addison's disease commonly react with the zona glomerulosa of adrenal cortex. We used high-resolution western blot analysis of an adrenal microsomal fraction to investigate the target of these antibodies. A protein with an apparent molecular weight of 54 kDa was recognised as the common and major component. Sera identifying this autoantigen (from 12 of 16 patients) also showed strong immunofluorescence staining of a steroid-producing human adrenal adrenocortical cell line, NCI-H295. On application of antisera specific for different cytochrome P450 steroidogenic enzymes (side-chain cleavage enzyme, 21-hydroxylase, 17 alpha-hydroxylase, 11 beta-hydroxylase) the mobility of the 54 kDa protein in western blots corresponded to that of 21-hydroxylase. This parallel behaviour was confirmed by immunoprecipitation, electrophoresis, and autoradiography with the various sera and 35S-methionine-labelled NCI-H295 cell lysates. Preabsorptions of 35S-methionine-labelled cell lysates with the antiserum to 21-hydroxylase, but not with the other enzyme antisera, abolished precipitation of the 54 kDa autoantigen with the patient sera. These results indicate that 21-hydroxylase (P450c21), prominent in the zona glomerulosa of the adrenal cortex, is a major autoantigen in idiopathic Addison's disease.