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*
Department of Medicine 1, Institutio Scientifico San Raffaele, Milan, Italy; and
Diabetes Research Institute and 3rd Medical Department, Krankenhaus München-Schwabing, München, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Studies in animal models of diabetes have shown that destructive autoimmunity is mediated through a Th1-dominated autoimmune response (10, 11, 12, 13). This has been suggested by some to arise relatively late in the disease process after a latent period of nonaggressive Th2 islet autoimmunity (8, 9). Other researchers have proposed that the natural course is characterized by spontaneous Th1 autoimmunity against a primary autoantigen with spreading to other islet Ags in the progression to diabetes (14, 15, 16). In both models, modulation of the autoimmunity to either maintain or initiate Th2 dominance, albeit by different strategies, impedes ß cell destruction (16, 17, 18, 19). Th1 and Th2 immunity in the mouse is associated with the generation of IgG2a and IgG1 Abs, respectively, and measurements of Ag-specific Ab subclasses have been used to identify periods of Th1- vs Th2-dominant immunity (20, 21, 22, 23, 24). In humans, the Ab responses of Th1 and Th2 immunity are not so distinct. There is some evidence that Th2 immunity is associated with IgG4 and IgE Ab responses, because both can be stimulated by the Th2 cytokine IL-4 (25, 26), and both are characteristic of the Th2-dominated response of atopy. The IgG subclass response found in Th1-dominated immunity appears inconsistent and can be IgG1 restricted or include most subclasses (27, 28).
A major limitation in our ability to characterize preclinical islet autoimmunity in humans is that subjects are usually identified when islet autoimmunity has already commenced, precluding the possibility of analyzing the early autoimmune response and subsequent changes in the progression to diabetes onset. To overcome this, we initiated the BABYDIAB study which investigates the temporal development of islet autoimmunity prospectively from birth in offspring of parents with diabetes (2). The study has shown that autoantibodies targeting insulin appear by 2 yr of age, and Abs to glutamic acid decarboxylase (GAD)3 and the protein tyrosine phosphatase IA-2 often appear simultaneously or soon after (29). In this paper, we have measured IgG subclass Abs to insulin, GAD, and IA-2 from initial autoantibody appearance until diabetes onset or current follow-up several years later in BABYDIAB offspring to determine whether there is regulation and change of autoantibody responses in the prediabetic period. The findings show heterogeneity, with an acute unregulated type of autoimmunity and rapid disease onset in some, and a slower "infectious" autoimmunity characterized by sequential IgG1-dominated autoantibody peaks to distinct islet autoantigens in others. Both support the presence of early destructive autoimmunity that may have the capacity for self-regulation. The infectious nature further suggests an ongoing Ag-specific regulation and spreading associated with periods of remitting and relapsing autoimmunity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BABYDIAB investigates the temporal development of islet autoimmunity from birth in offspring of parents with diabetes. It schedules regular visits with venous blood sampling at birth (cord blood), and at 9 mo and 2, 5, and 8 yr of age for measurements of autoantibodies to islets (ICA), insulin (IAA), GAD (GADA), and IA-2 (IA2A), and HLA class II typing. Since 1989, 2079 newborns have been recruited (1598 from a parent with type 1 diabetes and 481 from mothers with gestational diabetes). A total of 1391 have had a 9-mo visit; 1019 a 2-yr visit, and 298 a 5-yr visit. Twenty-six offspring have developed Abs to more than one islet autoantigen (median age of first islet autoantibody detection, 1.4 yr; range, 0.5–5.4 yr) and were included in this study. All 26 were followed with approximately yearly blood samples until the development of diabetes in 11 offspring (median onset age, 3.6 yr; range, 1.3–8.5 yr) or current age (median, 4.5 yr; range, 1.3–8.3 yr). In all offspring with elevated Ab levels repeated oral glucose tolerance tests were performed to monitor for elevated blood glucose levels. Type 1 diabetes diagnosis was defined according to World Health Organization criteria.
Autoantibody determination
IAA were measured by binding to 125I-labeled insulin in protein A/G and polyethylene glycol (PEG) competition radio-binding assays as previously described (29, 30, 31). IgG Abs to GAD and to IA-2 were measured by binding to [35S]methionine-labeled in vitro translated recombinant human Ag as previously described (29, 32). The upper limit of normal values was defined by the 99th percentile of Ab levels in nondiabetic control children (50 nU/ml IAA for the PEG assay and 4 units for the protein A/G assay, 3 units for GADA, and 5 units for IA2A).
IgG subclass and isotype autoantibodies
For IAA, GADA, and IA2A, the protein A/G radio-binding assays were used, substituting the addition of the protein A/G Sepharose with IgG subclass or isotype specific Ab bound Sepharose beads. Biotin-labeled mouse mAbs against human IgG1 (cat. no. 35052D), IgG2 (cat. no. 35072D), IgG3 (cat. no. 35082D), IgG4 (cat. no. 35092D), IgM (cat. no. 34152D), IgA1/IgA2 (cat. no. 35112D), IgE (cat. no. 34612D), and as a control for nonspecific binding against rat IgM (cat no. 10062D) were obtained commercially (PharMingen, San Diego, CA). Sepharose 4B streptavidin beads (Zymed, San Francisco, CA) were prepared by washing once with ice-cold PBS (50 mM phosphate buffer and 150 mM NaCl (pH 7.4)), followed by incubation of beads with biotinylated mAb with rotation at 4°C for 18 h, washing twice in PBS, once in assay buffer, and resuspension in assay buffer. The quantity of antiserum and streptavidin beads required to completely capture Ig in the assay reaction was determined by checkerboard titration under assay conditions. For IAA, this corresponded to 10 µl of Sepharose 4B streptavidin beads with 10 µg of Ab per well of the assay reaction, and for GADA and IA2A, 10 µl and 5 µg of Ab per well in the assay reaction.
IgG subclass specific IAA were measured by incubating 5 µl of serum with 1159 nU 125I-labeled insulin (Hoechst, Frankfurt, Germany; specific activity, 360 µCi/µg) in 25 µl 50 mM Tris and 1% Tween-20 (pH 8) (TBT) at 4°C for 72 h before addition of 50 µl Ab-coated Sepharose bead suspension, incubation for 1 h at room temperature, washing in cold TBT, and counting for 10 min. For each serum, nonspecific binding was also measured using beads coated with anti-rat IgM mAb. Results were expressed as nU insulin bound/ml of serum calculated as: [(IgG subclass specific counts - anti-rat IgM counts)/(total counts per tube)] x 1159 x 200.
All samples from the same offspring were tested for all subclasses in
the same assay. For each subclass the results from BABYDIAB offspring
showed two populations. One had a relatively normal distribution around
0 nU/ml ranging from -250 to +250 nU/ml, and a second above 250 nU/ml.
The threshold for each IgG subclass IAA was therefore placed at 250
nU/ml, which corresponded to 
200 counts after subtraction of the rat
IgM control. No control subjects had levels above 250 nU/ml.
For GADA and IA2A, 0.5 µl of serum was incubated with 25,000 cpm of [35S]methionine-labeled in vitro transcribed and translated Ag in 25 µl of 50 mM Tris, 150 mM NaCl, and 1% Tween 20 (pH 7.4) (TBST) overnight at 4°C, before addition of 50 µl Ab-coated Sepharose bead suspension, incubation for 1 h at room temperature, washing in cold TBST, and counting. Results were expressed as delta cpm (IgG subclass or isotype specific cpm - anti-rat IgM cpm) and converted to a SD score (SDS) calculated as: [(IgG subclass or isotype-specific counts delta cpm - mean delta cpm of control subjects)/SD delta cpm of control subjects]. The mean delta cpm + SD of 44 control subjects was used as the threshold for detection for each of the subclasses and isotypes tested. For GADA this was: IgG1, 71; IgG2, 47; IgG3, 41; IgG4, 55; IgM, 122; IgA, 57; and IgE, 68; and for IA2A: IgG1, 47; IgG2, 33; IgG3, 46; IgG4, 106; IgM, 72; IgA, 115; and IgE, 22. None of the control subjects had values above 3 SDS for any of the subclass or isotype GADA or IA2A. The specificity of the IgG subclass and isotype specific methods were validated using the MICA4 human IgG1 GAD mAb (33). SDS for IgG2, IgG3, IgG4, IgM, IgA, and IgE GADA measurements of the MICA4 supernatant and of MICA4 supernatant diluted 1/5 in normal human serum ranged between -1.5 and 1.6.
HLA typing
HLA-DR and -DQ alleles were determined using PCR-amplified DNA and nonradioactive sequence-specific oligonucleotide probes (34).
Data analysis
Linear regression analysis was used to correlate total and IgG subclass Ab responses. Kaplan-Meier life-table analysis was used to determine the cumulative risk of progression to type 1 diabetes. For all statistical methods the Statistical Package for Social Sciences (SPSS, Chicago, IL) was used.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Considerable changes in Ab titers for IAA, GADA, and IA2A were
found. In each offspring, and for each autoantibody, a peak titer was
reached soon after first detection (see examples in Fig. 1
). In most cases, decline was marked and
rapid after this peak. Peak responses to each Ag often occurred
sequentially in an almost infectious manner, suggesting continuous
regulation and spreading to new determinants. In case 1032, sequential
peaks in GADA, IAA, and IA2A were found, whereas in case 1649 the peak
sequence was IAA, GADA, IA2A. In case 1032 diabetes onset was
concurrent with a second rise in Abs to insulin and IA-2. In case 4005,
all Abs rose simultaneously with rapid diabetes onset. Case 1872 was
unique, developing both IAA and ICA with simultaneous peaks, but no
subsequent responses to either GAD or IA-2, and developing diabetes
when Abs had reached almost undetectable levels.
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In each of the cases shown in Fig. 1
, the peak responses
corresponded to major rises in the IgG1 Abs (Fig. 1, B–D).
This result was a consistent finding in all offspring. The magnitude of
peaks and decline of Ab and specifically IgG1 responses was often very
large, ranging from 184 to 7773 nU/ml (37–99% decrease; median, 90%)
for IAA, from 5 to 67 SDS (19–99% decrease; median, 73%) for GADA,
and from 3 to 124 SDS (35–99% decrease; median, 59%) for
IA2A.
Fig. 2 shows the age and sequence of IgG1
peak responses for each offspring. The median age of the first IgG1
peak for any of the Abs was 2.2 yr (interquartile range, 2–2.9 yr). A
first peak response to insulin (15 cases) was more frequent than to GAD
(6 cases, p < 0.01) or IA-2 (3 cases,
p < 0.001). The IgG1-IAA peak preceded other islet Ab
peaks in 13 offspring and was concomitantly first with a peak response
to GAD in one case (4204) and to IA-2 in another (1088). In five
offspring a peak response to GAD was found before IAA, and in one of
these concomitantly with an IA2A peak (1032). In the remaining six
offspring the peak sequence could not be determined: in five offspring,
the highest Ab titers to each Ag were found in the last sample obtained
before onset of diabetes which in all five was before age 4 yr, and in
one offspring (3975) only one sample with islet Abs has been tested. In
two offspring a second rise in the total and IgG1 Ab response to either
insulin (1032) and/or IA-2 (1032, 1088) was found before diabetes
onset. Of interest, in case 1032 the first IA2A peak was due to Abs
against epitopes in the juxtamembrane region of IA2, whereas in the
second IA2A peak the Abs were against distinct epitopes in the IA-2
tyrosine phosphatase domain (35).
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Distribution of autoantibody isotype and IgG subclass in prediabetes
For IAA, GADA, and IA2A, total Ab titers correlated with IgG1 Ab
levels (r = 0.8, 0.73, and 0.57, respectively; Fig. 3), but only slightly or not at all with
IgG2, IgG3, and IgG4 Ab levels (data not shown). Highest levels for all
Abs were seen for IgG1 (Fig. 4). For IAA,
IgG1 responses were frequently accompanied or followed by rises in
other IgG subclass responses, but for GADA and IA2A, other subclass
responses were less frequently found. In particular, IgG4 were often
elevated during the response to insulin (Fig. 4A) and in
four offspring (4005, 5006, 4050, 4215) IgG4-IAA levels were as high or
higher than IgG1-IAA (Table I). Low
levels of IgG4-GADA were found in only five offspring, and no offspring
had IgG4-IA2A. Table I shows which subclasses were detected in each
offspring. Seven offspring had IAA of all subclasses, one GADA of
all subclasses, and none IA2A of all subclasses. Exclusively IgG1
Abs were found for IAA in eight offspring, for GADA in eight offspring,
and for IA2A in nine offspring. None, however, had exclusively IgG1 Abs
to all three Ags, and with the exception of few cases, the subclass
response to one Ag was not usually paralleled with similar subclass
usage to other Ags. An exception is case 4204 who had IgG1, IgG3, and
IgE Abs to GAD and IgG1, IgG2, IgG3, and IgE Abs to IA-2. Two offspring
(4050, 4215) with strong and dominant IgG4 responses to insulin had
relatively high IgG2 titers to both GAD and IA-2. Of note is offspring
1948 in whom IgA-GADA was the first and major autoantibody
response.
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In most offspring there were minimal or no changes in the IgG
subclass islet Abs detected, with either a consistent presence of IgG1
responses only or the detection of additional subclass responses only
when IgG1 levels were at their peak. In a few cases, however, there was
later recruitment of other subclasses that did not parallel IgG1. These
are shown for IAA in Table II. Most
noticeably is the appearance of IgG4-IAA after first detection of
IgG1-IAA, and a persistence of the IgG4 response after significant
declines in the IgG1-IAA titer (1032, 1872, 2223, 6226, 6637). This was
also found for IgG2-IAA in three offspring (1032, 4204, 6637). In these
examples, IgG2-IAA were most similar to IgG4-IAA responses, whereas
IgG3-IAA paralleled the IgG1-IAA response. The development of IgG4-IAA
and their persistence was not associated with similar changes in the
subclass response to GAD or IA-2. Moreover, the predominant subclass Ab
against GAD or IA-2 did not change over time in all offspring.
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Diabetes onset usually occurred concurrent with a rise in islet Ab
titer. In five offspring diabetes developed at 1.8, 2.4, 3.2, 3.3, and
3.8 yr when all detectable islet Abs were at their highest levels.
Another four offspring had an early peak Ab response to one or more Ags
before diabetes, but either had peak responses to other Ags (4849,
4262, 1032) and/or second peak responses to the same Ags (1032, 1088)
at diabetes onset. Only two offspring (1872, 1085) developed diabetes
without rising Ab titers and after significant decline. Neither the
presence or absence nor the titers of specific IgG subclass Abs were
associated with progression to diabetes. A change to, or
persistence of, an IgG4-IAA response was not associated with protection
from diabetes. Specifically, cases 4005 (see Fig. 1) and 1872 (see Fig. 1 and Table II) developed diabetes when IgG4-IAA levels were at their
peak and higher than IgG1-IAA, cases 1032 (see Fig. 1 and Table II) and
3941 (data not shown) developed diabetes with rising IgG4-IAA, and case
4161 (data not shown) with persistent high levels of IgG4-IAA. By life
table analysis, risk of diabetes progression was elevated in offspring
with an exclusively IgG1 response to IA-2 compared with offspring who
had other subclass Abs to IA-2 (diabetes risk by age 5 yr, 56% (95%
CI: 24–88) vs 0%, p = 0.03), but exclusively IgG1
responses against insulin or GAD were not associated with rapid
diabetes development.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The consistent early burst of Ab activity, with peak responses to at least one Ag seen by 2–3 yr of age was a surprising finding. The relatively homogeneous timing of this appearance is consistent with programmed spontaneous islet autoimmunity in these children. Neonates acquire their ability to produce IgG responses after birth, are comparatively hypogammaglobulinaemic in the first months of life, and do not mount effective responses until late into the first year of age (39). The islet autoantibody responses found in the BABYDIAB offspring correlate well with this physiological maturation and may therefore be a visible expression of a defect in tolerance during immune repertoire formation. An alternative to defective tolerance is that islet autoimmunity in the offspring is a consequence of early ß cell damage. Maternal Ab is unlikely to cause damage because only a minority of those with maternally transferred Ab develop autoimmunity, and islet autoimmunity is at least as frequent in offspring of fathers with type 1 diabetes. Damage through environmental agents is possible, especially as the neonatal period is marked by a high susceptibility to infection (39). Increased infection in animal models, where insulitis appears uniformly already at around 4 wk of age (40), is however associated with protection from diabetes. Moreover, the relative absence of an initial IgM response to autoantigen in the offspring more likely speaks against direct immunization induced as a result of ß cell damage.
Although the timing of Ab appearance was homogeneous, the Ab sequence was not. In some there was an acute simultaneous autoantibody response to all or most diabetes-associated autoantigens. These offspring developed diabetes very early in life, suggesting a rapid unregulated form of destructive autoimmunity. Only some of these had the high risk HLA DRB1*03/DRB1*04 genotype. In the majority of offspring, however, Ab responses occurred in a wave of sequential peaks to distinct Ags. The first peak most frequently included IgG1-IAA. In a minority an IgG1-GADA peak preceded the IAA peak, whereas in most offspring IgG1-IA2A peaks followed, suggesting IA-2 as a secondary autoantigen target. The changes in Ab levels seen were striking, with a significant decline in Ab titer after peak if disease did not occur. This wave of Ab responses suggests either a correlate with Ag expression/presentation and/or regulatory mechanisms. In general, only one Ab peak to each Ag was found, and sequential peaks were against new distinct Ags when Ab response to previous Ags was declining. This is evident in case 1032 even at the epitope level, where two distinct Ab responses to IA-2 four to five years apart were against diverse epitopes (35). These data might argue for an induction of regulation which is Ag-specific, with subsequent, almost infectious, spreading of the autoreactivity to other Ags. Dampening of Ab titer could be achieved either by specific regulatory T cells (41) or may merely reflect exhaustion and apoptosis of autoreactive cells to early Ag (42) with spreading to new specificities.
It has been discussed whether IgG subclasses correlate with Th1 and Th2 immune responses. Some consider the early spontaneous autoimmunity in animal models of diabetes nondestructive with a Th2 component (8, 9), whereas others suggest that it is typically a Th1 autoreactivity from initiation (15). Regardless of the phenotype of initial autoimmunity, diabetes onset is most likely to be concurrent with ß cell destruction. In the BABYDIAB offspring, diabetes onset per se and thus ß cell destruction often occurred during an early or subsequent IgG1-dominated peak Ab response, suggesting that the IgG1 islet autoantibody response in these BABYDIAB offspring is characteristic of destructive autoimmunity. Because autoimmune ß cell destruction associated with type 1 diabetes is thought to be Th1 mediated, it follows that Th1 islet autoimmunity includes the production of IgG1 autoantibodies. This finding would be consistent with the subclass responses seen in infections inducing Th1 immunity (27). In this study we also show that the subclass distribution of the initial peak Ab responses is similar to that seen at diabetes onset, suggesting that a destructive, potentially Th1 autoreactivity may be present from initiation in these offspring.
The strongest evidence for an Ab subclass response being characteristic of a Th phenotype in humans is the link between IgG4 and Th2 immunity. IgG4 shares features of the Th2-associated mouse IgG1 and is stimulated by the Th2 cytokine IL-4 (25, 26). In the BABYDIAB offspring, IgG4 responses were most frequently found against insulin, IgG4-IAA detected in around half of the offspring. In some of these, IgG4-IAA was clearly not synchronous with IgG1-IAA, supporting the possibility that they are influenced by different factors. In four offspring, IgG4-IAA were the predominant IAA from initial detection, suggesting the presence of concurrent Th2 autoimmunity. The observation that persistent IgG4-IAA appeared after peak IgG1-IAA titers in a minority of offspring also suggests a recruitment of, or switch to, Th2 immunity. Importantly, however, the presence of or switch to IgG4-IAA, even when dominant, was not associated with protection from diabetes. Moreover, the detection of IgG4-IAA was not followed by an IgG4 response to GADA and/or IA-2, indicating that if IgG4 is a marker of Th2 autoimmunity in these offspring, then this phenotype exhibits Ag specificity.
IgG2 and IgG3 Abs to all three Ags could be detected, but were usually of low titer, and particularly for IgG3, found predominantly when IgG1 Ab levels were at their peak. Indeed, when detected, IgG3 responses generally paralleled IgG1, whereas IgG2 levels followed the IgG4 response in some individuals. The IgG1 dominance seen for GADA and IA-2A is consistent with previous reports examining ICA at onset of diabetes (43, 44), whereas IgG4-IAA have been reported (45). The subclass distribution of GADA found in BABYDIAB offspring differs, however, from that recently reported for older first degree relatives, where using ELISA for Ab measurement, IgG2- and IgG4-GADA were frequently found in the absence of IgG1-GADA, particularly in relatives who had not progressed to disease (24). Although the discrepancies could be explained by differences in the cohorts tested and methods used for Ab measurement, they are also suggestive of eventual switching in the subclass distribution of GADA. We found no evidence of a deviation away from an IgG1-dominated GADA response over time in BABYDIAB offspring, but we cannot exclude such changes in individuals with long-standing islet autoimmunity.
Diabetes onset occurred during a peak Ab response in most of the offspring who developed disease. The only other correlate to diabetes development was a significantly higher progression in offspring who had exclusively IgG1 Ab responses to IA-2, suggesting that a subclass restricted, potentially Th1-biased autoreactivity to late Ag could represent a more aggressive pathology. Changes in the subclass of autoantibodies did not correlate with protection or progression, and unlike what was reported for GADA (24), there was no evidence for slower progression if IgG2 or IgG4 autoantibody responses could be detected within the BABYDIAB offspring. Because IgG4 autoantibody responses may reflect an influence by IL-4, and a defect in the number and quality of IL-4 secreting NK-T cells together with low serum IL-4 levels has been reported in diabetes (46, 47), it will be relevant to measure serum IL-4 concentration in offspring before and after the development of autoimmunity to determine whether IL-4 concentrations are a better marker for disease protection.
The findings reported in the BABYDIAB offspring reflect the early islet autoimmunity associated with childhood type 1 diabetes in genetically susceptible individuals. We cannot predict whether autoimmunity arising later in life or in subjects with no family history of diabetes will be different, nor how the autoantibody response will behave in those BABYDIAB offspring who have autoimmunity but do not develop diabetes in childhood. Of interest is the IgA-dominated GADA response in offspring 1948 who developed islet autoimmunity relatively late. This indicates first, that other nonspontaneous forms of islet autoimmunity are likely, and second, that some of these forms could involve the mucosa.
In conclusion, the study of islet autoantibody titer, specificity, subclass, and previously epitope spreading in the BABYDIAB offspring cohort shows a very dynamic and active autoimmunity within the early pre-clinical phase of diabetes. We suggest that the relatively homogeneous timing of initial autoantibody responses in these offspring could be consistent with a spontaneous development of autoimmunity, and that this autoimmunity and the primary target may be genetically determined. The initial autoantibody response has characteristics of an early destructive autoimmunity which either results in rapid progression to disease or proceeds to a chronic autoimmune disease with remitting and relapsing activity.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Anette-G. Ziegler, Diabetes Research Institute, Koelner Platz 1, D-80804 Munich, Germany. E-mail address:
3 Abbreviations used in this paper: GAD, glutamic acid decarboxylase; IAA, insulin autoantibodies; GADA, GAD Abs; IA-2, insulinoma-associated cDNA 2; IA2A, IA-2 Abs; SDS, SD score; IDDM, type 1 insulin-dependent diabetes mellitus; ICA, ijlet cell Abs.
Received for publication February 1, 1999. Accepted for publication April 22, 1999.
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H. E. Naserke, E. Bonifacio, and A.-G. Ziegler Prevalence, Characteristics and Diabetes Risk Associated with Transient Maternally Acquired Islet Antibodies and Persistent Islet Antibodies in Offspring of Parents with Type 1 Diabetes J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4826 - 4833. [Abstract] [Full Text] [PDF]
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S. Trembleau, G. Penna, S. Gregori, G. Magistrelli, A. Isacchi, and L. Adorini Early Th1 Response in Unprimed Nonobese Diabetic Mice to the Tyrosine Phosphatase-Like Insulinoma-Associated Protein 2, an Autoantigen in Type 1 Diabetes J. Immunol., December 15, 2000; 165(12): 6748 - 6755. [Abstract] [Full Text] [PDF]
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 J. Luborsky, B. Llanes, R. Roussev, and C. Coulam Ovarian antibodies, FSH and inhibin B: independent markers associated with unexplained infertility Hum. Reprod., May 1, 2000; 15(5): 1046 - 1051. [Abstract] [Full Text] [PDF]
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L. Yu, D. T. Robles, N. Abiru, P. Kaur, M. Rewers, K. Kelemen, and G. S. Eisenbarth Early expression of antiinsulin autoantibodies of humans and the NOD mouse: Evidence for early determination of subsequent diabetes PNAS, February 15, 2000; 97(4): 1701 - 1706. [Abstract] [Full Text] [PDF]
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) and IA2A (
) and to nU/ml for IAA (•). For subclasses,
the y-axis corresponds to nU/ml of IAA subclasses
(B) or SDS of GADA (C) or IA2A
(D) subclasses. IDDM indicates type 1 diabetes
onset.
), GADA (
), and IA2A
(
