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Original Studies |
Department of Clinical Immunology and Allergy, Istituto di Semeiotica Medica (C.B., M.V., B.P.), University of Padova, Padova, Italy; FIRS Laboratories (S.C., B.R-S., J.F.), RSR Ltd., Cardiff CF4 5DU, United Kingdom; and Department of Medicine University of Wales College of Medicine (B.R-S., J.F.), Cardiff GF4 4XN, United Kingdom
Address all correspondence and requests for reprints to: Corrado Betterle, Istituto di Semeiotica Medica, University of Padova, via Ospedale Civile 105, Padova, Italy 35128. E-mail: betterle{at}ux1.unipd.it
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsRESULTSDiscussionReferences |
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-hydroxylase, and P450 side chain cleavage enzyme
were measured by immunoprecipitation assay using
35S-labeled recombinant proteins.
Adrenal-cortex autoantibodies and autoantibodies to 21-hydroxylase were
found in 81% of the patients with autoimmune Addison’s disease. None
of the patients with nonautoimmune Addison’s disease had
adrenal-cortex autoantibodies or autoantibodies to 21-hydroxylase. A
high association between these two markers in patients with different
forms of autoimmune Addison’s disease and in those with short- or
long-standing disease was found. Steroid-producing cells autoantibodies
were found in 26% of the patients with autoimmune Addison’s disease,
and autoantibodies to 17-hydroxylase and/or P450 side chain cleavage
enzyme in 36% of the patients. Steroid-producing cells autoantibodies
were found in 11/13 (85%) of patients with idiopathic premature
ovarian failure associated with autoimmune Addison’s disease, and
autoantibodies to 17-hydroxylase and/or P450 side chain cleavage
were found 12/13 (92%) of patients; the only case negative for all
these three markers suffered from Turner’s syndrome.
Provided that a high standard of immunofluorescence technique is maintained, measurement of adrenal cortex autoantibodies or steroid-producing cells autoantibodies by either immunofluorescence or immunoprecipitation assay is essentially equivalent.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsRESULTSDiscussionReferences |
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Two different immunoprecipitation assays (IPAs) for the detection of 21-OH autoantibodies (21-OH Abs) have been developed, one based on 35S-labeled 21-OH produced in an in vitro transcription-translation system (6, 9), the other based on 125I-labeled recombinant 21-OH produced in yeast (8).
These IPAs are highly specific and sensitive, and a good consistent agreement between results of 21-OH Abs measurement using these assays and ACAs by IIT was found (6, 7, 8). However, one report from a different laboratory suggested no agreement between ACAs and 21-OH Abs results mainly in patients with long-standing autoimmune AD (10).
The IIT on cryostat sections of testis and/or ovary has until recent years been the only method available for the detection of steroid-producing cells autoantibodies (StCAs) (1). In patients with autoimmune AD, StCAs generally correlate with the presence of primary gonadal failure (hypergonadotropic hypogonadism) (1, 11). In the absence of primary gonadal failure, StCAs can herald the future development of the disease (12, 13).
35S-Labeled recombinant antigens have also been used for
the detection of 17-hydroxylase autoantibodies (17-OH Abs) and
P450 side chain cleavage enzyme autoantibodies (P450scc Abs) (7). In
this preliminary study a close association between StCAs detected by
IIT and 17-OH and P450scc Abs assayed by IPA was demonstrated (7).
In contrast, others studies indicated that there were some
discrepancies between StCAs and 17-OH and P450scc Abs measurement
mainly in patients with APS type 1 (4, 14, 15, 16, 17).
The aim of this study was to investigate the prevalence of ACAs and
StCAs by IIT and 21-OH Abs, 17-OH Abs, and P450scc Abs by IPA in a
large cohort of patients with different forms of autoimmune and
nonautoimmune AD with or without idiopathic hypergonadotropic
hypogonadism and the associations between these markers.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsRESULTSDiscussionReferences |
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We studied 165 Italian patients with primary clinical AD: 143
were affected by autoimmune AD and 22 were affected by nonautoimmune
AD. In the autoimmune AD group, 21 patients had APS type 1 (mean age
12.1 yr, mean duration of the disease 8.1 yr, range 0–30 yr), 55
patients had APS type 2 (mean age 31 yr, mean duration of the disease
7.4 yr, range 0–46 yr), 67 patients had isolated AD (mean age 23.6,
mean duration of the disease 4.8 yr; range 0–30 yr). Thirteen females
were affected by idiopathic premature ovarian failure (POF) (12 with
autoimmune POF and 1 with Turner’s syndrome). With regard to the
duration of the disease, data were collected from 125 patients with
autoimmune AD, from 62 patients with a disease duration of 
2 yr
(short-standing disease), and 63 patients with a disease duration >2
yr (long-standing disease). In the group of patients with nonautoimmune
AD, 10 patients had disease due to tuberculosis, 9 had
adrenoleukodystrophy, 2 had adrenal insufficiency due to primary
adrenal neoplasia, and 1 had congenital AD due to enzyme defect. All
sera were coded and tested blindly for ACAs, StCAs, 21-OH-Abs, 17-OH
Abs, and P450scc Abs. The correlation between ACA titers and 21-OH Ab
levels was calculated in 85 patients after logarithmic transformation
of 21-OH Ab levels and ACA titers.
Indirect IITs
ACAs were tested by the classical IIT using thin cryosections of normal human adrenal tissue (2, 3). Positive sera were retested by doubling dilution to the end point by the same method. StCAs were tested by the indirect complement fixation test using thin cryosections of normal human ovarian tissue (13).
IPAs
35S-labeled 21–0H, 35S-labeled
17-OH, and 35S-labeled P450scc were produced in an
in vitro transcription/translation system (Promega Corp., Southampton, UK), and the labeled proteins used in IPAs
were as described previously (6, 7) to test 21-OH Abs, 17-OH Abs,
P450scc Abs reactivity of each serum.
Statistical analysis
The statistical significance of the associations between
autoantibodies detected by different methods was determined by
-square test. Correlation between ACA titers and 21-OH Ab levels was
calculated after logarithmic transformation of ACA titers and
21-OH.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsRESULTSDiscussionReferences |
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In patients with autoimmune AD, ACAs were found in 116/143 (81%)
patients and 21-OH Abs in 116/143 (81%) patients. All 22 patients with
nonautoimmune AD were negative for ACAs or 21-OH Abs (Fig. 1a
). Prevalence of ACAs and 21-OH Abs
varied in the three different groups of patients with autoimmune AD
(Fig. 1b). In patients with short-standing disease, the prevalence of
ACAs was 90% (56/62) and that of 21-OH Abs 92% (57/62); in patients
with long-standing disease, the prevalence of ACAs was 79% (50/63) and
that of 21-OH Abs 78% (49/63) (Fig. 1c). The relationship between the
two tests is shown in Fig. 1d. Sera from 155/165 (94%) patients were
concordant in the two assays and sera from 10 patients showed
discrepant results (5 were low positive for ACAs but negative for 21-OH
Abs and 5 were low positive for 21-OH Abs but negative for
ACAs).
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(r = 0.85).
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-OH Abs, and/or P450scc Abs
StCAs were found in 37/143 (26%) patients with unselected
autoimmune AD and 17-OH and/or P450scc Abs were found in 51/143
(36%) of patients. All were also positive for ACAs and 21-OH Abs. Of
22 subjects with nonautoimmune AD, only one patient in the
adrenoleukodystrophy group was positive for P450scc Abs at low levels;
all were negative for StCAs and 17-OH Abs (Fig. 3a). The prevalence of StCAs and 17-OH
and P450scc Abs greatly varied in the different patient groups with
autoimmune AD as shown in Fig. 3b. StCAs were found in 11/13 (85%) of
patients with idiopathic POF associated with autoimmune AD, and
17-OH and/or P450scc Abs in 12/13 (92%) of the patients. StCAs were
also present in 26/130 (20%) patients without POF, whereas 17-OH
and/or P450scc Abs were present in 39/130 (30%) of this non-POF group
(Fig. 3c). All but one of the 26 StCA-positive patients were also
positive for 17-OH and/or P450scc Abs. The association between these
assays is shown in Figure 3d: 148/165 (90%) sera were concordant in
the two assays, 16 sera showed discrepant results (15 were low positive
for 17-OH Abs and/or P450scc Abs but negative for StCAs and 1 was
positive for StCAs and negative for 17-OH Abs and/or P450scc). StCAs
were significantly associated with 17-OH and/or P450scc Abs in
patients with APS type 1 (P = 0.001) and 2
(P < 0.001), isolated autoimmune AD (P
< 0.001), and POF (P < 0.0001).
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shows the immunological
combinations in the 13 patients with idiopathic POF associated with AD.
The patient affected by Turner’s syndrome was the only case negative
for all the three autoantibodies.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsRESULTSDiscussionReferences |
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-OH, and
P450scc have been recognized as the main autoantigens in adrenal and
ovarian autoimmunity (1). Previous studies from our laboratory have
shown good agreement between ACAs detected by IIT and 21-OH Abs
detected by IPA (6, 7, 8) not confirmed by others mainly in patients with
long-standing disease (10). The present report on a large number of sera (n = 165) from patients with different forms of AD demonstrates unequivocally that ACAs and 21-OH Abs measurements are closely associated, with 94% of the sera showing concordant results in the two assays. Only 6% of the sera gave discrepant results but were, in general, at a low titer. A strong association between ACAs and 21-OH Abs was recently confirmed in a follow-up study on 58 patients with organ-specific autoimmune diseases without overt hypoadrenalism (2, 3). Furthermore, during the follow-up, all ACAs and 21-OH Abs-positive children developed overt adrenal failure (3); in the case of the adults, all of the 12 patients who progressed to clinical AD during the follow-up were positive for both ACAs and 21-OH Abs (2).
The current study is in good agreement with our previous observations (2, 3, 6, 7, 8) and confirms in a larger number of patients that ACA or 21-OH Ab measurements are valuable markers for identifying patients with autoimmune AD in different forms (APS type 1 and 2 and isolated), in different stages (potential, subclinical, and clinical), and with different durations of the disease.
The reasons for the discrepancy between our observations and results reported by Falorni et al. (10) may be related to the demanding technical aspects of the ACA determination by IIT rather than to the selection of patients or to the duration of autoimmune AD. Furthermore, the current study shows that measurement of ACAs by IIT or 21-OH Abs by IPA is essentially equivalent and that they remain the best markers for the identification of the patients with autoimmune AD (clinical, subclinical, or potential).
Idiopathic POF can be associated with autoimmune AD, but its prevalence is quite different in the various forms of the disease. It is often associated with APS type 1, whereas it is less frequent in APS type 2 and quite rare in patients with isolated autoimmune AD (19). StCAs are well-established markers of POF associated with autoimmune AD but are quite rare in other forms of POF (1, 11, 20).
In the present study we showed that in patients with autoimmune AD the
frequency of StCAs greatly varied, but they are highly associated with
measurements of autoantibodies to 17-OH Abs and/or P450scc Abs,
which have been identified as the main gonadal autoantigens (7). In
patients with idiopathic POF in the context of autoimmune AD, StCAs,
and 17-OH and/or P450scc Abs must be considered good markers of
autoimmune POF. The only patient with autoimmune AD and idiopathic POF
negative for StCAs, 17-OH Abs, and P450scc Abs disclosed a
nonautoimmune POF. However, in patients with autoimmune AD without POF
there were 16 discrepant results between the two tests. In particular,
sera from 15 patients were positive for 17-OH Abs and/or P450scc Abs
but negative for StCAs (Fig. 3d
). This may reflect a greater
sensitivity of the IPA based on 35S-labeled recombinant
autoantigens produced in the in vitro
transcription/translation system.
Moreover, in the case of autoimmune AD without POF, StCAs have been
demonstrated to be good markers of potential autoimmune POF only in
females (12, 13). Patients with adrenal autoimmunity without POF who
are positive for 17-OH Abs and/or P450scc Abs in the absence of
StCAs may also be at risk for developing POF. A further follow-up study
of such patients should be helpful to assess the role of 17-OH Abs
and/or P450scc Abs alone in the natural history of the POF (13).
The autoimmune AD and POF appear to be diseases mediated by cytotoxic T
lymphocytes. ACAs and 21OH Abs and StCAs, 17-OH Abs, and P450scc Abs
are likely to be mere markers of the autoimmune process. The study of
the epitopes recognized by ACAs and 21-OH Abs was unable to
differentiate patients with different forms of autoimmune AD or
potential AD (21). Only the identification of the autoepitopes
recognized by autoreactive T lymphocytes infiltrating the adrenal
cortex and the ovary could improve our knowledge of the pathogenesis of
this disorder.
Overall, our studies indicate that ACAs and 21-OH Abs are
characteristic of the four different forms of autoimmune ADs (APS type
1, APS type 2, isolated, and potential) and StCAs and autoantibodies to
17-OH and/or P450scc appear to be good markers of POF associated
with adrenal autoimmunity. ACA and StCA measurements are demanding
technically but provided that this is recognized, the measurement of
adrenal autoantibodies by either immunofluorescence or
immunoprecipitation assays is essentially equivalent.
Received July 15, 1998.
Revised October 28, 1998.
Accepted November 3, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsRESULTSDiscussionReferences |
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-hydroxylase. Lancet. 339:770–773.[CrossRef][Medline]
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