Constant, but not pulsed calcitriol suppresses hemodialysis patients' antigen-induced lymphocyte proliferation.

BACKGROUND/AIMS: In vitro constant calcitriol [1,25-(OH)(2)D(3)] inhibits healthy individuals' T lymphocyte proliferation at supraphysiological concentrations. In contrast, among hemodialysis patients, intravenous 1,25-(OH)(2)D(3) pulse therapy of secondary hyperparathyroidism has been shown to be even immunostimulatory. We studied the effect of in vitro constant and intermittent 1, 25-(OH)(2)D(3) on lymphocyte antigen response of hemodialysis patients. METHODS: Twelve hemodialysis patients' peripheral blood mononuclear cells were stimulated with purified protein derivative of tuberculin (12.5, 25 and 50 mg/l) or tetanus toxoid (TT; 1,000, 5, 000 and 10,000 Lf/l, limit of flocculation) for 7 days. Constant 1, 25-(OH)(2)D(3) was added to all cultures at concentrations of 0, 10(-10) or 0.25 x 10(-9) mol/l (0, 42 and 105 ng/l) and to half of the cultures additionally as a 0.75 x 10(-9) mmol/l (315-ng/l) pulse on the 5th culture day. RESULTS: TT-induced lymphocyte proliferation was statistically related to a constant 1,25-(OH)(2)D(3) concentration (p = 0.001, analysis of variance). With constant 1, 25-(OH)(2)D(3) concentrations of 0, 42 and 105 ng/l, the TT-induced responses were 1.53, 1.44 and 1.40 log cpm, respectively (mean of TT concentrations). The responses of the (additionally) pulse-treated cells [1.65, 1.50 and 1.40 log cpm; concentrations of constant 1, 25-(OH)(2)D(3) as above] were similar to those of the nonpulsed cells. Thus constant, but not pulsed 1,25-(OH)(2)D(3) decreased the TT responses. On the purified protein derivative of tuberculin response, neither constant nor pulsed 1,25-(OH)(2)D(3) had any significant effect. CONCLUSIONS: The decline of TT response with constant 1,25-(OH)(2)D(3) corresponds with findings on immunosuppressive action of 1,25-(OH)(2)D(3) in previous studies done on normal subjects' cells. This was not seen with intermittently applied 1,25-(OH)(2)D(3). These results support the previous concept that intermittent 1,25(OH)(2)D(3) therapy is not immunosuppressive in hemodialysis patients.

Adrenal autoimmunity: results and developments.

Autoimmune Addison's disease (autoimmune adrenalitis) often occurs in autoimmune polyendocrinopathy syndromes APS1 (APECED) and APS2. Although the genetic background and etiology of the two syndromes is remarkably different, they both result in a similar autoimmune destruction of the adrenal cortex. Recently, the defective gene in APS1, AIRE (autoimmune regulator) was identified, whereas in APS2, the major genetic factor remains to be found in the human major histocompatibility complex haplotype (HLA) region. In addition to the genetic factors, the recent findings in genetics and immunity leading to the pathogenesis of adrenal autoimmunity in polyendocrinopathy syndromes are discussed.

RNA and protein expression of the murine autoimmune regulator gene (Aire) in normal, RelB-deficient and in NOD mouse.

Mutations in the putative transcription factor autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiosis-ectodermal dystrophy (APECED; OMIM#240300), a monogenic recessively inherited disease characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiosis of mucous membranes and ectodermal dystrophies. In this study the expression of murine homolog for AIRE protein, Aire, was detected in a fraction of thymic medullary epithelial cells. Subcellularly, in thymus the protein appears as concentrated into nuclear dot-like structures, whereas in transfected cells the protein is also bound along a cytosolic fibrillar network. By RT-PCR Aire mRNA was detected in thymus, lymph node, spleen and testis although the second round PCR amplified Aire specific band from most mouse tissues analyzed. Furthermore, the Aire mRNA was detected in dendritic cell (DC) populations isolated from thymus and spleen, representing both myeloid- and lymphoid-related lineages of DC. We also demonstrate that the Aire protein is absent in the thymus of RelB-deficient mouse and in NOD thymus most of the Aire positive cells showed an abnormal morphology. These results suggest that the Aire protein is associated with the normal development and/or action of a subset of thymic medullary stromal cells involved in tolerance induction.

3beta-hydroxysteroid dehydrogenase autoantibodies are rare in premature ovarian failure.

Premature ovarian failure (POF) is a disorder of heterogeneous etiology, and autoimmunity has been suspected as one cause of POF. The steroidogenic enzyme, 3beta-hydroxysteroid dehydrogenase (3betaHSD), has been characterized as a potential autoantigen in POF as well as in insulin-dependent diabetes mellitus (type 1 diabetes). Here we studied the presence of steroid cell antibodies (SCA), autoantibodies to 3betaHSD and to two other known autoantigens in ovarian failure, steroidogenic enzymes 17alpha-hydroxylase (P450c17), and side-chain cleavage enzyme (P450scc) in POF patients and patient groups with autoimmune polyendocrinopathy syndromes type 1 and 2 (APS1 and -2), isolated Addison's disease, type 1 diabetes, and healthy controls. The SCA were found in 2 of 48 POF, 11 of 15 APS1, and 1 of 9 APS2, and autoantibodies to in vitro translated 3betaHSD protein were detected in 1 POF serum associated with Addison's disease and 3 APS1 sera. All 3betaHSD precipitating sera were also positive for SCA. However, no SCA or 3betaHSD autoantibodies were found in 38 Addison's disease, 28 type 1 diabetes, and 71 healthy control sera. In analysis of autoantibodies to P450c17 and P450scc, antibodies to these enzymes were not found in POF sera, but were found in 10 and 12 APS1 patient sera, respectively, and 1 APS2 patient serum contained anti-P450c17 antibodies. Our results show that autoantibodies to 3betaHSD in POF patients are rare and are also found in patients with APS1.

Immunochemical characterization and taxonomic evaluation of the O polysaccharides of the lipopolysaccharides of Pseudomonas syringae serogroup O1 strains.

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) of Pseudomonas syringae pv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear alpha-D-rhamnan with the tetrasaccharide O repeat -->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-R hap-(1-->2)- alpha-D-Rhap-(1--> (chemotype 1A). The same alpha-D-rhamnan serves as the backbone in branched OPSs with lateral (alpha1-->3)-linked D-Rhap, (beta1-->4)-linked D-GlcpNAc, and (alpha1-->4)-linked D-Fucf residues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the lateral D-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular left arrow over right arrow branched irregular --> linear OPS structure alterations (1Cleft arrow over right arrow 1C-1A --> 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D left arrow over right arrow 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a(1), Ps1a, Ps1a(1), and Ps1a(2), as well as MAbs Ps1b, Ps1c, Ps1c(1), Ps1d, Ps(1-2)d, and Ps(1-2)d(1) specific to epitopes related to the lateral sugar substituents of the OPSs, were produced against P. syringae serogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme of P. syringae was improved. Screening with MAbs of about 800 strains representing all 56 known P. syringae pathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS of P. syringae and related pseudomonads as a phylogenetic marker is discussed.

Isolation and characterization of the mouse Aire gene.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a rare autosomal recessive disorder characterized by Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have other clinical symptoms both within and outside the endocrine system, mainly as a result of autoimmunity against organ-specific autoantigens. The gene for APECED has recently been identified and termed AIRE (for AutoImmune REgulator). APECED is a model of organ-specific autoimmunity and isolation and characterization of the homologous mouse gene, Aire, will provide tools for dissection of the mechanisms underlying this human disorder and defining molecular pathways involved in organ-specific autoimmunity. We have isolated and completely sequenced the mouse Aire gene which is split into 14 exons over 13 kb and encodes a predicted protein of 552 amino acids. The predicted mouse and human AIRE proteins are 71% identical and contain motifs suggestive of a transcriptional regulator. Additional conserved motifs are emerging in the AIRE/Aire proteins including a nuclear localization signal, an "ASS" domain, and a "SAND" domain. The human and mouse AIRE promoters have conserved sites for several thymus-specific transcription factors and others important in hematopoesis, consistent with its expression in rare cells of the thymus medulla, lymph nodes, and fetal liver. We have mapped mouse Aire to mouse chromosome 10 by FISH, to the same region as Pwp2 and Pfkl, confirming synteny to the corresponding region of human chromosome 21.

A common mutation in Sardinian autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients.

Autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED; also called APS-1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is characterized by two of the three major clinical symptoms that may be present: Addison's disease, and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. We have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes and fetal liver, and encodes a protein with two putative zinc fingers and other motifs suggestive of a transcriptional regulator. Seven mutations have been described to date, including R257X, the predominant Finnish and northern Italian APECED allele, which has also been observed in other patients of diverse origin on different haplotypes. A 13-bp deletion (1094-1106del) has also been observed in several patients of different geo-ethnic origin. The other described mutations appear to be rare. We present mutational analyses of the AIRE gene in ten Sardinian APECED families and show that there is a mutation, R139X, associated with one predominant haplotype unique to the Sardinian patients (18/20 independent alleles). The carrier frequency of R139X in Sardinia is 1.7%, giving an estimated population frequency of APECED of 1/14,400. Using linkage disequilibrium data, the estimated age of the R139X mutation is between 20 and 25 generations. A previously described 13-bp deletion was also observed on an allele of one patient. The identification of a single common Sardinian APECED mutation will facilitate its genetic diagnosis. Given the carrier frequency of R139X in the Sardinian population, AIRE may be implicated in the pathogenesis of other autoimmune diseases in the Sardinian population, particularly those affecting the endocrine system.

Common mutations in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy patients of different origins.

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED; OMIM *240300, also called APS 1,) is a rare autosomal recessive disorder that is more frequent in certain isolated populations. It is generally characterized by two of the three major clinical symptoms that may be present, Addison's disease and/or hypoparathyroidism and/or chronic mucocutaneous candidiasis. Patients may also have a number of other clinical symptoms including chronic gastritis, gonadal failure, and rarely, autoimmune thyroid disease and insulin-dependent diabetes mellitus. We and others have recently identified the gene for APECED, which we termed AIRE (for autoimmune regulator). AIRE is expressed in thymus, lymph nodes, and fetal liver and encodes a protein containing motifs suggestive of a transcriptional regulator, including two zinc finger motifs (PHD finger), a proline-rich region, and three LXXLL motifs. Six mutations, in cluding R257X, the predominant Finnish APECED allele, have been defined. R257X was also observed in non-Finnish APECED patients occurring on different chromosomal haplotypes suggesting different mutational origins. Here we present mutation analyses in an extended series of patients, mainly of Northern Italian origin. We have detected 12 polymorphisms, including one amino acid substitution, and two additional mutations, R203X and X546C, in addition to the previously described mutations, R257X, 1096-1097insCCTG, and a 13-bp deletion (1094-1106del). R257X was also the common mutation in the Northern Italian patients (10 of 18 alleles), and 1094-1106del accounted for 5 of 18 Northern Italian alleles. Both R257X and 1094-1106del were both observed in patients of four different geo-ethnic origins, and both were associated with multiple different haplotypes using closely flanking polymorphic markers showing likely multiple mutation events (six and four, respectively). The identification of common AIRE mutations in different APECED patient groups will facilitate its genetic diagnosis. In addition, the polymorphisms presented provide the tools for investigation of the involvement of AIRE in other autoimmune diseases, particularly those affecting the endocrine system.

Structure of the O polysaccharide and immunochemical relationships between the lipopolysaccharides of Pseudomonas syringae pathovar tomato and pathovar maculicola.

O polysaccharides (OPS) of the lipopolysaccharides (LPS) of Pseudomonas syringae pathovar tomato GSPB 483 and pathovar maculicola IMV 381 were studied by 1H-NMR and 13C-NMR spectroscopy, including two-dimensional COSY, rotating-frame NOE spectroscopy (ROESY), and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The OPS from both strains were shown to be chemically identical. Two structurally different types of repeating units (O repeats 1 and 2) were elucidated in each OPS. The minor O repeat is a pentasaccharide having the structure 2. The same structure has been reported earlier for some other OPS of P. syringae. The major O repeat is a hexasaccharide with the novel structure 1 which is distinguished from 2 by the presence of the second lateral residue of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) and by a different position of substitution of one of the L-rhamnose (Rha) residues in the backbone. [structures: see text] Structural and serological diversity of OPS of strains belonging to P. syringae pv. tomato and pv. maculicola and phylogenetic relatedness of the strains are discussed.

Structural heterogeneity in the O polysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W-43).

The O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W-43) was studied by Smith degradation and 1H-NMR and 13C-NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, and H-detected 1H,13C heteronuclear multiple-quantum coherence (HMQC) experiments. The OPS was shown to consist of pentasaccharide O repeats of two types both containing four L-rhamnose and one 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc) residue. Structure 1 of the major O repeat which had been established earlier [Das, S., Ramm, M., Kochanowski, H. & Basu, S. (1994) J. Bacteriol. 176, 6550-6557], was confirmed by our data, and a new structure 2 was elucidated for the minor O repeat and found to differ from the structure 1 only in the position of substitution of one of the rhamnose residues in the main chain. [structures: see text] A role of structural and immunochemical features of the LPS for defining the taxonomical position of the bacterium studied is discussed.

Positional cloning of the APECED gene.

Autoimmune polyglandular syndrome type I (APS 1, also called APECED) is an autosomal-recessive disorder that maps to human chromosome 21q22.3 between markers D21S49 and D21S171 by linkage studies. We have isolated a novel gene from this region, AIRE (autoimmune regulator), which encodes a protein containing motifs suggestive of a transcription factor including two zinc-finger (PHD-finger) motifs, a proline-rich region and three LXXLL motifs. Two mutations, a C-->T substitution that changes the Arg 257 (CGA) to a stop codon (TGA) and an A-->G substitution that changes the Lys 83 (AAG) to a Glu codon (GAG), were found in this novel gene in Swiss and Finnish APECED patients. The Arg257stop (R257X) is the predominant mutation in Finnish APECED patients, accounting for 10/12 alleles studied. These results indicate that this gene is responsible for the pathogenesis of APECED. The identification of the gene defective in APECED should facilitate the genetic diagnosis and potential treatment of the disease and further enhance our general understanding of the mechanisms underlying autoimmune diseases.

Interleukin-10 gene expression induced by HIV-1 Tat and Rev in the cells of HIV-1 infected individuals.

The role of cytokines in the regulation and function of the immune system is of great importance. In human immunodeficiency virus (HIV) infection, with progressive deterioration of cell-mediated immune response, cytokines are dysregulated. We have therefore investigated cytokine mRNA expression in type-1 and type-2 helper T cells of HIV-seropositive (HIV+) individuals, stimulated with mitogen (leukoagglutinin) and HIV-1 Tat and Rev peptides, previously found to induce proliferative T-cell responses in these individuals. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect interleukin 2 (IL-2), interferon gamma (IFN-gamma), IL-4, and IL-10 mRNAs. There was no difference in the mRNA expression of these cytokines when the cells of HIV-infected or noninfected individuals were polyclonally stimulated with the mitogen, as all cytokine mRNAs were detected in both groups. Baseline cytokine expression of unstimulated cells was, however, different in these two groups: the cells of HIV+ persons did not show comparable expression of mRNAs to HIV-seronegative (HIV+) individuals. When the cells of HIV+ individuals were stimulated with the peptides, 70% of the cases showed IL-10 mRNA expression, 20% IFN-gamma, and 10% IL-2, with no detection of IL-4 mRNA in any of the cases. Our results thus show that HIV-specific T-cell antigens induce production of IL-10 in HIV-infected individuals. The increase in IL-10 demonstrated here may have a role in hyperactivation of B cells, as well as in immunosuppression of T cells often seen in HIV-infected individuals.

Detection of candidal antigens in autoimmune polyglandular syndrome type I.

Autoimmune polyglandular syndrome type I (APS I) is associated with chronic mucocutaneous candidiasis. To characterize the antibody responses in this subgroup of Candida albicans infections, we screened a candidal cDNA expression library with patient sera and found four cDNA clones encoding the immunopositive proteins enolase, heat shock protein 90, pyruvate kinase, and alcohol dehydrogenase. The reactivity to these antigens was studied further by immunoprecipitation assays with in vitro-transcribed and -translated proteins. Analysis of sera from 44 APS I patients showed that the highest antibody reactivity was found with enolase (80% of patients reactive), but significant serological responses were also found with heat shock protein 90 (67%), pyruvate kinase (62.5%), and alcohol dehydrogenase (64%). Overall, 95.5% of patients had detectable antibodies to at least one of these proteins. The cDNAs of enolase and heat shock protein 90 were also expressed in Escherichia coli and studied by immunoblotting. Again, 84% of sera reacted with enolase, whereas 44% of sera reacted with heat shock protein 90. A good correlation between the two methods was found for both enolase (r = 0.86; n = 58; P < 0.001) and heat shock protein 90 (r = 0.71; n = 56; P < 0.001). Our results indicate that the four abundant candidal proteins are the major antigens and can be used as accurate markers of candidiasis in APS I patients. The immunoprecipitation assay described here is particularly useful for the rapid analysis of a large number of samples.

Helper and cytotoxic T cell responses of HIV type 1-infected individuals to synthetic peptides of HIV type 1 Rev.

In cell-mediated immunity T cells recognize peptide fragments of the antigenic protein in association with major histocompatibility complex (MHC) proteins. Synthetic 9- to 16-mer peptides have been widely used to identify the region(s) of a protein that act as T cell epitope. Here, we report antigenic peptides identified on HIV-1 regulatory protein Rev. Four synthetic peptides (amino acids 9-23, 25-39, 33-48, and 41-56) were first shown to stimulate T helper (Th) cell proliferation in peripheral blood lymphocytes (PBLs) derived from HIV-seropositive (HIV+) individuals. The same peptides induced cytotoxic T lymphocyte (CTL) activities toward the autologous target cells incubated with the peptides. Both responses were specific to the HIV infection as HIV-seronegative (HIV-) control individuals showed no significant proliferative or cytotoxic activity. The proliferating cells were CD4+ T cells, and CTL activity was mediated by CD8+ human leukocyte antigen (HLA)-restricted T cells. The identification of peptides containing epitopes that can induce both Th and CTL responses to regulatory proteins of HIV-1 in infected individuals might be important for vaccine development against AIDS. Since early regulatory proteins of HIV are expressed by the infected cells before the initiation of the synthesis of structural proteins, a CTL response against these proteins could destroy the infected cells before the release of infectious virions.

Mapping of B cell epitopes on steroid 17 alpha-hydroxylase, an autoantigen in autoimmune polyglandular syndrome type I.

Earlier studies have shown that 17 alpha-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc) are the main autoantigens recognized by sera from patients with Addison's disease associated with autoimmune polyglandular syndrome type I (APS I). In this study we tried to identify the autoantigenic epitopes on P450c17 and compared the identified sequences with corresponding regions in two other adrenal autoantigens, P450scc and P450c21. A series of P450c17 cDNA fragments was expressed in Escherichia coli and the recognition of the corresponding protein fragments by patient sera was tested by immunoblotting. Four distinct epitope regions (ER) were found: ER1 (amino acids 122-148), ER2 (280-304), ER3 (396-432) and ER4 (466-508). B cell epitope prediction analysis showed that the four identified ERs were all located in regions of high predicted antigenicity. Homology search revealed that ER3 and ER4 but not ER1 and ER2 were related to similar sequences on P450c21. No significant homologies with P450scc in these regions were found. Although all three P450 cytochromes are genuine autoantigens this finding suggests that the autoantibody reaction against P450c17 and P450c21 can partly be a result of immunological cross-reactivity.

Characterization of adrenal autoantigens recognized by sera from patients with autoimmune polyglandular syndrome (APS) type I.

Steroidogenic enzymes P450scc, P450c17 and P450c21 have recently been shown to be the main autoantigens recognized by sera from patients with autoimmune polyglandular syndrome (APS type I) with or without Addison's disease. We have studied the interrelationships of autoantigens revealed with APS type I sera in the adrenal and in placenta by immunodiffusion and immunoblotting, and correlated the findings to reactivities towards the above steroidogenic enzymes. We studied 50 patients with APS type I, 36 of whom also had Addison's disease, three patients with isolated (adult type) Addison's disease, seven healthy relatives of the patients with APS type I, 18 patients with insulin-dependent diabetes mellitus, 17 patients with autoimmune liver disease and 26 healthy controls. Immunodiffusion revealed two precipitating autoantigens in adrenal gland, and one of these was also found in placenta. In immunoblotting, five major adrenal antigens with molecular sizes of 55 kDa, 48 kDa, 43 kDa, 39 kDa and 19 kDa were seen. Reactivity to the 55 kDa, 39 kDa or 19 kDa represents the occurrence of antibodies to P450c17 or to its components as revealed by comparative studies with mouse antibodies to recombinant P450c17. These three bands in immunoblot as well as precipitating antibodies were observed exclusively in APS type I patients who had or developed Addison's disease. The 48 kDa antigen was found also in placenta and is probably P450scc, judged by the high correlation of P450scc antibodies with immunodiffusion and immunoblotting results using placental homogenate. There was no association between reactivity to the 43 kDa band and other immune parameters studied. The results thus indicate that P450scc and P450c17 enzymes are the precipitating adrenal autoantigens recognized by sera from APS type I patients. However, according to immunoblot results there could be some yet unidentified adrenal autoantigens to which APS type I patients could develop antibodies.

Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison's disease.

Patients with idiopathic Addison's disease have autoantibodies reacting with adrenal cortex. If Addison's disease is associated with other endocrine immune diseases like autoimmune polyglandular diseases (APD) type I and type II, antibodies may recognize all steroid-producing cells. We showed previously that one antigen recognized by APD-I sera is the cytochrome P450c17 hydroxylase. We have now looked for antibodies to P450c17 and to two other key enzymes in the steroid biosynthetic pathway, the P450scc and P450c21, in a series of patients with isolated Addison's disease (8 patients) or with APD-I or APD-II (50 and 9 patients, respectively). The result of antienzyme antibodies were further correlated with the immunofluorescence pattern against adrenal gland, testis, ovary, and placenta, and with the clinical findings presented. In APD-I patients with Addison's disease and in APD-II patients, antibodies to at least one of the P450 enzymes were frequently found (positive findings in 81% and 78%, respectively). Such antibodies were less frequent in APD-I patients without Addison's disease (21%) and in the isolated Addison cases (25%). In APD-I, antibodies recognized as frequently P450c17 and P450scc, specific for all steroid-producing cells as the adrenal specific enzyme P450c21. In contrast, patients with APD-II or with the isolated Addison's disease reacted almost exclusively with P450c21. Immunofluorescence studies showed good correlation with the known fact that the zona glomerulosa of the adrenal cortex is devoid of the P450c17, that the Leydig cells of the testis and the theca interna cells of the ovary express P450c17 and P450scc, and that the placental trophoblasts express only P450scc. The presence of antibodies to P450scc or to at least one of the tested P450 enzymes correlated significantly to gonadal failure in the females but not in the males.