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Department of Bacteriology and Immunology (M.G., E.K., L.K., A.M.), University of Helsinki, FIN-00014 Helsinki, Finland; Department of Immunology (M.G., R.V., L.K., M.-L.S., A.M.), Helsinki University Central Hospital Laboratory Diagnostics, FIN-00290 Helsinki, Finland; Department of Surgery (L.H.), Helsinki University Hospital, FIN-00014 Helsinki, Finland; National Veterinary and Food Research Institute (S.S.), FIN-00581 Helsinki, Finland; Department of Molecular Medicine (M.H.), National Public Health Institute, FIN-00300 Helsinki, Finland; Department of Medical Science (O.K.), University Hospital, SE-75185, Uppsala, Sweden; and The Hospital for Children and Adolescents (M.H., J.P.), University of Helsinki, FIN-00290 Helsinki, Finland
Address all correspondence and requests for reprints to: Aaro Miettinen, M.D., Ph.D., Department of Bacteriology and Immunology, Haartman Institute, P.O. Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland. E-mail: aaro.miettinen{at}helsinki.fi.
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Abstract |
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Introduction |
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In organ-specific autoimmune diseases, the patient typically has circulating autoantibodies specific for the affected organ. Often these precede the clinical disease by years and persist for years after its onset. This is also true for most of the autoimmune components of APECED (7, 8). However, convincing evidence has not been presented for parathyroid autoimmunity. Autoantibodies against the parathyroid gland (PG) epithelia (9, 10, 11, 12, 13, 14, 15) and endothelia (16) have been described but are controversial (17). No confirmed parathyroid autoantigens are known, but PTH antibodies have been described (18), and recently, evidence (19) was presented for the extracellular domain of the Ca2+-sensing receptor (CaSR) being an autoantigen in IHP, including APECED.
Pathogenetic mechanisms other than autoimmunity have also to be considered. AIRE is expressed in the medullary epithelium of the thymus (20). The thymus and the PGs originate from the third pharyngeal pouches via shared organ primordia under control of shared transcription factors (21, 22). Mice deficient in the transcription factors Crkol or Tbx1 develop coexisting hypoplasia of the thymus and the PGs (23, 24). Also, in mice, the thymus produces PTH and expresses CaSR (25), and PTH may be produced in human thymomas (26). Thus, there is a close relationship between the thymic and parathyroid epithelia. If AIRE is expressed in PGs, its lack might cause their hypoplasia and/or increased sensitivity to insults.
With our large series of patients with APECED, we have evaluated sex and HLA class II as factors determining the occurrence of HP. With regard to pathogenetic mechanism, we have searched for parathyroid autoantibodies and studied whether the human PGs express AIRE mRNA.
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Subjects and Methods |
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The criterion for the diagnosis of APECED was the presence of two of HP, AF, and chronic or recurring mucocutaneous candidiasis, or one of these if a sibling had at least two. This study includes all the 90 Finnish patients for whom we had the required data by the end of year 2001; 45 were males and 45 females. AIRE mutations have been identified for 65 of them. The major Finnish mutation 769C>T was present in 55.
Serum samples taken in 1972 through 2001 were available from 63 of the patients, stored aliquoted at -20 C. Samples taken within 1 yr of the diagnosis of HP were available from 16 of the patients. We studied the last available sample from the patients without HP and one to five samples from each patient with HP.
The diagnosis of HP was based on persistent hypocalcemia and hyperphosphatemia in the absence of renal failure. PTH levels were not analyzed, because our previous studies showed that the levels in the HP patients were under the detection limit (Perheentupa, J., unpublished observations).
As controls, we used sera from blood donors, children subjected to elective surgery for unrelated reasons, and children with newly diagnosed type 1 diabetes (27). The Ethical Committees of the hospitals approved the study.
Sex as a determinant
To evaluate sex as determinant of the phenotype, we calculated, for the whole series of patients, sex specific incidences of the commonest endocrine components HP and AI over the age intervals of 0–1.99, 2.00–4.99, 5.00–9.99, 10.00–14.99, 15.00–19.99, 20.00–29.99, and 30.00–39.99. From the incidences, we estimated the prevalences at the end of these intervals.
HLA determinations
Association between HP and HLA class II genes was searched for by studying the HLA-DRB1, DQA1, and DQB1 alleles (8) from 55 index patients, 11 of whom did not have HP.
Tissues
Human hyperplastic PGs, from patients with chronic uremia, we obtained from the Department of Surgery; and human thymic tissue, removed at open heart surgery, was from the Hospital for Children and Adolescents. Dog PGs were from animals exterminated for unrelated reasons at the National Veterinary and Food Research Institute, Helsinki. The tissues used for immunofluorescence (IF) studies were snap-frozen in isopenthane cooled with liquid nitrogen. For isolation of PG fractions or total RNA, the tissues were processed immediately (see below) or frozen in liquid nitrogen and stored at -70 C.
Anti-PG antibodies
For the indirect IF studies, we used, as substrates, cryostat sections (5-µm thick) of canine or human PGs. Tissue sections, fixed in acetone at -20 C for 10 min, were incubated with the sera diluted 1:2 and 1:10 in PBS, pH 7.2, at room temperature for 30 min, washed three times (each 10 min) with PBS, and reacted with the secondary antibody for 30 min. After washing, the slides were mounted with Immumount (Shandon Inc., Pittsburgh, PA). Positive sera were tested further at 5-fold dilution steps. Fluorescein isothiocyanate (FITC)-labeled rabbit antihuman IgG (Dako A/S, Glostrup, Denmark), diluted 1:300, was used as the secondary antibody. An Olympus BX 50 microscope, equipped with interference filters for FITC, was used for microscopy. For documentation of the results, we used Kodachrome 800/1600 color slide film, or an Orca IIIm CCD-camera (Hamamatsu Photonics, Hamamatsu City, Japan) and Openlab version 2.2.3 software for Macintosh (Improvision, Coventry, UK).
For the Western blotting experiments, human PG tissue was homogenized in sucrose [85.75 mg/ml (250 mM)] made in HEPES buffer [0.238 mg/ml (10 mM)], pH 7.4, with added protease inhibitors phenylmethylsulfonylfluoride [174.2 µg/ml (1 mM)], pepstatin A [1 µg/ml (1.458 µM)], antipain [1 µg/ml (1.654 µM)], and EDTA [2.606 mg/ml (7 mM)], on ice, and centrifuged at 600 gmax at 4 C for 10 min. The supernatant was collected and recentrifuged at 100,000 gmax at 4 C for 60 min. The pellet (membrane fraction) was homogenized in PBS with protease inhibitors. The suspended pellet and supernatant (soluble fraction) were aliquoted in reducing sample buffer, boiled for 5 min, and stored at -70 C or used immediately. The proteins were separated by SDS-PAGE (8% or 15% gels) under reducing conditions and transferred to nitrocellulose sheets. For immunoblotting, the membrane strips were first incubated with PBS containing 3% BSA (BSA-PBS) and 0.02% NaN3, at +37 C for 2 h, to block nonspecific binding sites. Serum samples were diluted 1:400 in 3% BSA-PBS containing 0.2% Triton X-100 (Fluka Chemi GmbH, Buchs, Switzerland) and incubated with the strips at 4 C overnight. After washing with PBS-0.2% Triton X-100, the strips were incubated with rabbit antihuman IgG (dilution, 1:5000; Dako) coupled with horseradish peroxidase, at room temperature for 2 h. After further washing, the bound antibodies were visualized using the enhanced chemiluminescence reaction (Amersham Life Science, Buckinghamshire, UK).
Anti-CaSR antibodies
We searched for CaSR antibodies by using a radioimmunoprecipitation assay as described for anti-GAD65 and anti-IA-2 antibodies (27). The human CaSR cDNA was kindly donated by Dr. M. Freichel (University of Heidelberg, Mannheim, Germany) (28). Its extracellular domains and the first membrane-spanning domain, 1949 base pairs, were ligated into the HindIII/SacI site of the pSP64 PolyA Vector (Promega, Madison, WI). The ligated vector was propagated in Escherichia coli JM 109 and purified by the Plasmid Midi Kit (QIAGEN GmbH, Hilden, Germany). A peptide containing amino acids 1–649 of the human CaSR protein was produced by in vitro transcription and translation of the purified plasmid using the TNT Coupled Reticulocyte Lysate System (Promega) in the presence of [35S]cysteine (Amersham, Little Chalfont, Buckinghamshire, UK), rather than [35S]methionine, for labeling, because the extracellular domain of CaSR has only five methionines but 21 cysteines. Unincorporated [35S]cysteine was removed by gel chromatography on NAP-5 columns (Amersham Pharmacia Biotech, Uppsala, Sweden). To establish the assay and to construct a standard curve, we used the rabbit antirat CaSR IgG cross-reacting with human CaSR (Affinity BioReagents, Inc., Golden, CO). The cut-off point for this assay was established at the mean + 3 SD of the level of 192 adult blood donors. The mean value used was recalculated after omitting the results of obvious outliers (binding > mean + 5 SD).
CaSR antibodies we also studied by immunoprecipitation of the labeled receptor with sera from patients or control subjects, followed by analysis of the precipitates by SDS-PAGE and autoradiography, as described by Li et al. (19).
Anti-PTH antibodies
PTH antibodies were searched for by both an ELISA and an immunoprecipitation technique. For the ELISA, 96-well polystyrene plates (Greiner Labortechnik, Germany) were coated first with varying amounts (1–3 µg/ml) and later with 1 µg/ml (0.106 µM) human PTH (Sigma, St. Louis, MO) in 100 µl PBS at +4 C overnight, washed 3 times with 0.05% Tween 20 (Fluka Chemie GmbH) in PBS (PBS-T), and incubated with 1% BSA in PBS-T at room temperature for 1 h. Rabbit antihuman PTH peptide (amino acids 39–84) antibodies (Biotrend Chemikalien GmbH, Köln, Germany), and preimmune rabbit serum in dilutions 1:100 to 1:10,000 or normal rabbit IgG in concentrations 0.5–5.0 µg/ml, were used to set up the assay, and later included in every ELISA plate to establish the standard curve. For anti-PTH antibody analysis, 100 µl patient or control sera, at dilution 1:100 in 1% BSA PBS-T, were added in the wells, incubated at room temperature for 2 h, and washed as above. Then 100 µl rabbit antihuman IgG (dilution 1:600; Dako) or swine antirabbit IgG (dilution 1:2,000; Dako) coupled with horseradish peroxidase was added in the wells and incubated at room temperature for 1 h. After further washing, 100 µl substrate solution (ABTS Single Solution; Zymed Laboratories Inc., San Francisco, CA) was added in the wells, and the absorbance was read at OD 405 nm. Each serum was analyzed in duplicate. The cut-off point for this assay was established at the mean + 3 SD of the level of 66 blood donors.
The radiobinding assay was modified from that of Williams et al. (29) for insulin autoantibodies. Human PTH 1–84 was labeled with 125I, using the Bolton-Hunter reagent (Amersham), as described by the manufacturer. The specific activity of the labeled peptide was 12 µCi/µg. In brief, 5 µl patient or control sera and 15,000 cpm [125I]PTH were added in 50 µl TBT buffer (50 mM Tris, pH 8.0, 1% Tween 20) and incubated on a shaker at 4 C for 72 h. Immune complexes were precipitated by adding 50 µl of a 20% suspension of protein A Sepharose CL-4B (Pharmacia Biotech) in TBT buffer and shaking at +4 C for 90 min. The resulting precipitates were washed four times in the precipitation buffer and counted in a 
-counter (1272 Clinigamma; Wallac, Turku, Finland). The results were expressed as reference units based on a standard curve constructed with serial dilutions (1:50 to 1:1250) of the rabbit antihuman PTH peptide antibodies for which an arbitrary RU value was assigned. The cut-off point for this assay was the mean + 3 SD of the level of 25 blood donors.
Detection of human AIRE mRNA
Total RNA was isolated from snap-frozen tissue samples homogenized in TRIzol Reagent (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. RNA samples were treated with deoxyribonuclease (Dnase RQ1, Promega) to eliminate contaminating genomic DNA. One microgram of total RNA was reverse-transcribed at 60 C for 30 min using oligo(dT)15 primers (Promega) and C. therm. Polymerase (Roche Diagnostics, Mannheim, Germany). For nested amplification of AIRE from cDNA, the following primers were used: sense (5'-GAG TTC TAC ACT CCC AGC AAG TTC-3') and antisense (5'-GAC TCC AGG TCA TCC CTG TG-3') designed to amplify exons 6–13 (868 bp) in the first round and sense (5'-AGC TCC ACC AGA AGA ATG AGG A-3') and antisense (5'-CAG TGA GTA CAC CGC AGC AC-3') primers amplifying exons 8–11 (477 bp) in the second. The amplification was carried out by denaturation at 94 C for 3 min, followed by 35 cycles at 94 C for 30 sec, at 62 C for 30 sec, at 72 C for 1 min, and a final extenuation at 72 C for 5 min in a vol of 25 µl containing 2 µl template, 2 µl 0.2 mM of each primer, 0.1 mM of each deoxynucleotide triphosphate in PCR buffer (Promega), and 0.125 U AmpliTaq DNA polymerase (Perkin-Elmer Cetus, Norwalk, CT). Thermal cycler PTC-200 (MJ Research, Watertown, MA) was used. Control amplification was performed with ß-actin primers using conditions as above, except using 30 cycles and annealing at 57 C. The PCR products were analyzed by agarose gel (1.5%) electrophoresis and ethidium bromide staining.
Statistical analysis
For statistical analysis, we used 
2 statistics with Yates’ correction, or Fisher’s exact test, as appropriate. All statistical analyses for the HLA associations were performed using the SPSS 10.0 package for Windows (30).
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Results |
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Of the 90 patients, 76 had HP, its prevalence being 71% for the males and 98% for the females (P < 0.001; see Fig. 1
for age-specific incidences and prevalences). The incidence was clearly higher for the whole group of female patients than male patients at the age range of 2–15 yr. Age at diagnosis varied for the female patients from 1.6–23 yr (median, 5.2 yr) and for the male patients from 1.6–43 yr (median, 7.5 yr). At the age of 15 yr, the prevalence was 94% for the females but only 59% for males (P < 0.001). HP was the first endocrinopathy of 51 patients, viz. 18 (40%) of the male patients and 33 (71%) of the female patients. Fourteen patients had no HP and, strikingly, 13 of them were men. Of these 13 non-HP males, eight were 28–54 yr old, or beyond the latest age of appearance of HP in the female patients, and three were 45–54 yr old, or beyond the latest age of appearance of HP in the males.
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HLA analysis was performed for 55 index patients, 11 of whom were non-HP. We compared the frequencies of DRB1* and DQB1* alleles in patients with and without HP. No statistically significant association was observed (data not shown).
Antiparathyroid antibodies
Using the indirect IF technique, we (13) and others (9, 10, 11, 12, 13, 14, 15) have detected, in patients with APECED, antibodies binding to human PG tissue. However these patients were later suggested to have human-specific antimitochondrial antibodies causing false positive IF results (17). To avoid this problem, we used canine PGs as substrates. Of the 63 patients tested, 12 (19%) had antibodies binding to canine PG epithelial cells (P < 0.001) (Fig. 2 and Table 1). Of the 14 samples taken from the patients within 1 yr of the diagnosis of HP, four (29%) were positive, but this was not different from the non-HP patients (Table 1). The antibody levels were low (titers 
50). Also, we tested some of the sera on cryostat sections of human hyperplastic PGs. Thirteen of 35 of the APECED patients (37%), five of 20 of the blood donors (25%), and four of 20 of the type 1 diabetes patients (20%) were positive (P > 0.05).
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and 4). Antigens of more than 200 kDa, and approximately 70 kDa, 60–65 kDa, 45–50 kDa, 32 kDa, 30 kDa, 18 kDa, 11 kDa, and 9 kDa were often recognized. Of the samples taken from the patients within 1 yr of the diagnosis of HP, 7 of the tested 15 bound to the more-than-200-kDa, 11 to the 60–65-kDa, 6 to the 45–50-kDa, 12 to the 32-kDa, 10 to the 18-kDa, 7 to the 11-kDa, and 4 to the 9-kDa proteins of the soluble or membrane fractions. The sera from patients with and without HP did not differ clearly in their reactions. In longitudinal analysis, series of samples taken from individual patients showed changes in reactivity, but these were not consistent (Fig. 4). IF positivity and binding to the more-than-200-kDa, 45-kDa, 32-kDa, and 18-kDa proteins showed some correlation.
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To detect anti-CaSR antibodies, we tested first the immunoprecipitation technique of Li et al. (19) and could demonstrate that some patient sera precipitated the radiolabeled CaSR, as shown by SDS-PAGE and autoradiography (Fig. 5). Also, some control sera gave a faint precipitate (results not shown). For the analysis of the sera from the patients and controls, we used the more quantitative immunoprecipitation technique (27). By this, seven (12%) of the 60 APECED patients tested, but also 8 of 192 of the blood donors (4%; P > 0.05) and 1 of 30 of the diabetic children (3%; P > 0.05), had precipitating antibodies (Table 1
). Only one of the 16 samples taken within 1 yr of the diagnosis of HP showed CaSR-antibodies.
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No AIRE mRNA in the parathyroids
By nested RT-PCR, AIRE mRNA could be demonstrated in the thymic tissues, but not in the hyperplastic human PG tissues tested (Fig. 6).
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Discussion |
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Interestingly, we observed a clear sex difference with regard to HP. Of our 90 patients, 14 had escaped developing HP and, strikingly, 13 of them were male. Also, the incidence of HP was clearly higher for the females than the males at the age of 2–14 yr (Fig. 1). The prevalence was, in the whole series, 71% for the males, in contrast to 98% for the females (P < 0.001) and, at the age of 15.0 yr, it was 59% and 94%, respectively (P < 0.001). The latest age of appearance of HP was 23 for the females, in contrast to 43 for the males. Of the 13 non-HP males, eight were 28–54 yr old, or beyond the latest age of appearance of HP in the female patients, and three were 45–54 yr old, or beyond the latest age of appearance of HP in the male.
No sex difference has been reported for the APECED phenotype, save a clearly higher incidence of primary gonadal failure in the female, which has been explained by protection of the testis by the blood-testis barrier (31). Female preponderance is common in autoimmune diseases, but it usually increases after puberty (32). Sex hormones may determine this difference, because estrogen enhances immune responses and androgens seem to decrease them (33). But the sex differences that we now discovered in APECED appear already in early childhood and can thus hardly be explained by sex hormones.
This sex difference in the occurrence of HP was clearly associated with a difference in the occurrence of AF. Of the 14 non-HP patients, 12 had AF, since the median of 17 yr ago. The first or only endocrinopathy was AF in 24 patients, viz. 21 (47%) of the male patients but only 3 (7%) of the females. Of these 24 patients, only 12 had HP (11 of 21 males and 1 of 3 females) vs. 18 expected from the prevalence of HP in the series. Thus, AF was associated with absence of HP in 12 patients, all but two of them male, eight beyond the female age range of appearance of HP, and two even beyond the male range, being in their mid-50s.
AF thus seems to protect against destruction of the PGs. APECED patients with AF as the first disease component other than candidiasis were reported to develop fewer disease components than other patients (5), and it now turns out to be especially true with regard to HP. Somehow, autoimmunity initiated against the adrenal cortex might result in down-regulation of the autoimmune process. Alternatively, cortisol substitution therapy might be the factor protective against further autoimmunity, because it cannot exactly reproduce the physiologic situation.
The HLA alleles DRB1*15 and DQB1*0602 protect the patients with APECED of type 1 diabetes, and DRB1*03 seems to be positively associated with the tendency to develop AF (30). Our present study of the DRB1, DQA1, and DQB1 alleles of 11 patients without and 44 patients with HP did not reveal any associations with HP.
The etiology of HP of APECED remains unknown. It differs from the congenital forms of HP by not occurring before the age of 1 yr (34). Because the other endocrinopathies of APECED are typical autoimmune diseases, it is natural to assume that this is also true of the HP. Alternatively, the lack of functional AIRE could affect the PG epithelia directly or indirectly by decreasing their resistance to endogenous or exogenous injuries. We explored these possibilities.
Some evidence for PG autoimmunity has been found. Lymphocyte infiltration of PG has been observed in patients (35) and in dogs with IHP (36) and in the autoimmune prone NOD mice without overt HP (37). Using leukocyte migration inhibition assay and human PG extracts, T cell reactivity was observed in patients with IHP (38), but also controversial results exist (39).
Autoantibodies against PG antigens have been described by several groups (9, 10, 11, 12, 13, 14, 15, 16, 18, 19). Using the indirect IF technique, we found antibodies against canine PG epithelia in 19% of the patients with APECED but in none of the control subjects. However, there was no difference in the frequency of antibodies between the patients with and without HP (Table 1), and the antibody levels were always low. Also, we detected antibodies binding to human hyperplastic PG epithelia, but not significantly more often than in the control subjects. Our results agree with the earlier reports of antiparathyroid antibodies and that nonspecific antibodies may disturb the assays using human PG tissue as a substrate. The patients may have antibodies against human mitochondria (17) or against other cellular autoantigens typical for inflammatory conditions or APECED (15, 40).
By Western blotting, antibodies binding to several PG proteins were observed in most sera. IF positivity showed some correlation with antibodies binding to proteins of more than 200 and approximately 45, 32, and 18 kDa. No systematic differences in the binding patterns were evident on comparison of the sera from the patients with and without HP. When the sera taken within 1 yr of the diagnosis of HP were compared with samples taken several years later, changes seen in the reactivity were not consistent (Fig. 4). Thus, it remains open whether any of the proteins detected by immunoblotting are relevant in the pathogenesis of HP.
The patients did not have antibodies against the extracellular domain of CaSR more frequently or in higher amounts than the control subjects. This is contradictory to Li et al. (19), who found antibodies against CaSR in 6 of 17 (35%) of patients with APECED, suggesting that the receptor is a PG autoantigen in APECED. Using their technique (19), we also found that some patient sera precipitated the labeled CaSR (Fig. 5). However, by the more quantitative immunoprecipitation assay (27), only 12% of the patients with APECED and 4% of the blood donors were positive. This difference is not significant (P > 0.05). In addition, none of the patients had antibody levels higher than some of the control subjects, suggesting that the precipitation reactions were not a sign of HP. Our results are in line with those of A. Söderberg et al. (Söderberg, A., A. G. Myhre, O. Ekwall, K. Gebre-Mehdin, H. Hedstrand, E. Landgren, A. Miettinen, P. Eskelin, M. Halonen, T. Tuomi, J. Gustafsson, E. S. Husebye, J. Perheentupa, M. Gylling, M. P. Manns, F. Rorsman, O. Kämpe, T. Nilsson, personal communication; University of Uppsala, Sweden), and P. Goldsmith and A. Spiegel (unpublished results; National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD), who could not find anti-CaSR antibodies in APECED. Söderberg et al. studied a larger series of patients, using a similar radioimmunoprecipitation technique as we and the same clone of the extracellular domain of the CaSR as antigen, whereas Goldsmith and Spiegel used an ELISA technique and the extracellular domain of human CaSR as antigen. A possible explanation to the discrepancy between our results and those of Li et al. (19) is that we used a more quantitative assay and tested larger series of patients and controls. We also tested the sera for antibodies against PTH. By ELISA, 3% of the APECED sera had antibodies binding to PTH, but so did also 3% of the blood donors. By the immunoprecipitation technique, we did not find any anti-PTH antibodies. Stimulating antibodies against the CaSR were described recently in rare patients with hypocalciuric hypercalcemia combined with other autoimmune manifestation (41). Thus, the CaSR may be an autoantigen, but our results suggest that neither the CaSR nor the PTH is an important autoantigen in the HP of APECED.
AIRE is normally expressed in the thymic medullary epithelial cells. The thymic and PG epithelia have a partly shared embryonic origin (21, 22, 23, 24). In mice, the thymus can even produce PTH (25). Whether this is true also in man is not known, but some thymomas produce PTH (26). Also, AIRE might be normally expressed in the PG epithelia, as it is in the thymic medullary epithelium, and its lack might sensitize the PGs to injuries. Human AIRE mRNA has been found in targets of autoimmunity, including adrenal cortex, testis, and thyroid (1), but this is controversial (2). The specific situation of the PGs is not known. Hence, we isolated total RNAs from hyperplastic human PGs, and we analyzed these for the expression of AIRE using nested RT-PCR techniques. We found expression of AIRE in thymic extracts but not in the PGs.
Lack of AIRE may affect the negative selection of T cells normally executed by the thymic medullary epithelial cells and thymic dendritic cells. The recent results obtained in aire-deficient mice support this hypothesis and suggest that aire is a transcriptional enhancer of tissue-restricted antigens in the medullary epithelial cells of the thymus (3, 4). Their ectopic expression is necessary for the clonal deletion of T-lymphocytes with high-affinity receptors for these peripheral antigens (42). It is tempting to speculate that, in APECED, some not-yet-identified parathyroid autoantigen(s) is not expressed in the thymic medullary epithelial cells. Consequently, dangerous autoreactive T cells are released into the circulation and, when activated, induce parathyroid autoimmune disease.
In conclusion, we found evidence for autoimmune etiology of the HP of APECED. Our results do not confirm the earlier report that the CaSR would be an important autoantigen in HP. Further work is needed for the identification of the relevant autoantigens and to explain why the parathyroids are affected so often in the patients lacking the functional products of the AIRE gene. The new interesting finding, that most of the patients without HP are males, shows that, in addition to HLA alleles, sex also has modifying effects on the phenotype of APECED.
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Footnotes |
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Abbreviations: AF, Adrenocortical failure; APECED, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy; CaSR, Ca2+-sensing receptor; FITC, fluorescein isothiocyanate; HP, hypoparathyroidism; IF, immunofluorescence; IHP, idiopathic HP; PBS-T, Tween 20 in PBS; PG, parathyroid gland.
Received April 21, 2003.
Accepted July 1, 2003.
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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70 kDa, 
