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Original Studies |
Department of Pediatrics (A.-M.H., M.S.R., M.K.), University of Oulu, FIN-90400 Oulu; Hospital for Children and Adolescents (H.K.Å., M.K.), University of Helsinki, FIN-00290 Helsinki; and Medical School, University of Tampere (M.K.), and Department of Pediatrics, Tampere University Hospital, FIN-33014 Tampere, Finland
Address all correspondence and requests for reprints to: Mikael Knip, M.D., Hospital for Children and Adolescents, University of Helsinki, P. O. Box 281, FIN-00290. HUCH, Finland. E-mail: mikael.knip{at}hus.fi
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We are entering a new era in the treatment of type 1 diabetes, with possible preventive actions that may represent the end of this complicated disease. Primary prevention covers all tools that aim at preventing the initiation of insulitis. Such strategies should be applied to the general population of children to be effective. Theoretically, this would be an ideal approach, but at present it is limited by lack of knowledge of which factors could be safely and easily modified early in life. At the moment most intervention trials are focused on secondary prevention in at-risk individuals in whom signs of ongoing ß-cell destruction have been detected by the occurrence of disease-associated antibodies in the peripheral circulation (8). To maximize the effectiveness of secondary prevention, the first screening should be as early as possible, and it has been suggested that screening should be initiated at birth (9).
Large scale screening of the general population must be cost-effective, which means that the number of samples and the costs per sample should be minimized. It has been suggested that the first line screening in the general population should be an analysis of antibodies to glutamic acid decarboxylase (GADA) and to the islet cell antigen IA-2 (IA-2A) in a single combined assay, followed by the determination of islet cell antibodies (ICA) in those subjects with detectable levels of either or both of the above antibodies (10). GADA have been detected at a lower frequency in children with type 1 diabetes diagnosed before the age of 10 yr, however, whereas insulin autoantibodies (IAA) have a high disease sensitivity in young children (10, 11). There is still no consensus regarding which antibody or antibody combination should be used in screening for signs of ß-cell autoimmunity in infants and toddlers.
Most of the type 1 diabetes-associated antibodies are of the IgG class and are actively transported through the placenta (12, 13); accordingly, newborn infants of mothers with type 1 diabetes have been shown to have insulin antibodies (IA), ICA, and other disease-associated antibodies in their circulation as a consequence of transplacental transfer (4, 14, 15, 16) (Hämäläinen, A.-M., et al. unpublished observation). Early screening thus generates the problem of differentiating between endogenously produced autoantibodies in the infant and maternally acquired antibodies. We set out to study the elimination of maternally acquired antibodies, the pattern of this elimination, and the time needed for them to disappear from the peripheral circulation in infancy.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The primary population comprised 136 mothers and their newborn infants from families with at least one first degree relative with type 1 diabetes who had entered the second pilot study of the Trial to Reduce IDDM in the Genetically at Risk (TRIGR) project in Finland, which was aimed at evaluating the possible effect of the elimination of cow’s milk proteins in early infancy on the risk of the infants of progression to type 1 diabetes later (17). The study design was double blind, and the infants were randomized into 2 groups. After exclusive breast feeding, those in the intervention group were given a casein hydrolysate formula (Nutramigen, Mead Johnson & Co., Evansville, IN) for the first 6–8 months of life, while the infants in the control group were given a regular formula (Enfamil, Mead Johnson & Co.) supplemented with 20% casein hydrolysate formula to make the 2 similar in smell and taste. The infants were born between March 1995 and December 1996 in 15 hospitals around Finland. A cord blood sample was taken for the analysis of antibodies associated with type 1 diabetes. The actual index population included those 47 infants (34.6%) whose cord blood samples were positive for 1 or more disease-associated antibodies. Thirty-eight of these (80.9%) were born to a mother with type 1 diabetes, 8 into a family with an affected father, and 3 into a family with an affected sibling. This includes 1 family in which the father and 1 of the siblings had type 1 diabetes and 1 in which both parents were affected. The mean age of the mothers at delivery was 30.4 ± 4.8 (±SD) yr (range, 20–44 yr). The severity of type 1 diabetes in the affected mothers was classified according to White (18); 10 subjects were class B, 9 were class C, 15 were class D, and 4 were class F. The mean duration of diabetes at delivery was 14.9 ± 8.0 yr (range, 1–34 yr). Mean individual hemoglobin A1C levels throughout pregnancy ranged from 4.6–7.4% (mean, 6.3 ± 0.7%), given a reference range of 4–6% in nondiabetic subjects (19). Two of the initially unaffected mothers had gestational diabetes treated with dietary therapy. One of the nondiabetic mothers had an oral glucose tolerance test performed during pregnancy, and the result was normal. Thirty-three of the infants were boys (68.1%). The mean gestational age was 38.2 ± 1.4 weeks (range, 35.1–42.6 weeks), mean weight at birth was 3830 ± 662 g (range, 2470–5240 g), and mean length was 50.4 ± 2.6 cm (range, 44.0–54.7 cm). In addition to the cord blood sample, blood samples were taken at follow-up visits at the ages of 3, 6, 9, 12, 18, and 24 months. A blood sample was taken from the mother at the time of delivery. The serum samples for the antibody assays were stored at -20 C until analyzed. All samples from each infant and the maternal sample obtained at delivery were analyzed in the same assay. Written informed consent was obtained from the mother before enrolment. The study was approved by the joint ethics committees of the participating hospitals.
ICA assay
ICA were determined by a standard immunofluorescence method using sections of frozen human group O pancreas (20). End-point dilution titers were examined for the positive samples, and the results were expressed in Juvenile Diabetes Foundation (JDF) units relative to an international reference standard. The detection limit was 2.5 JDF units. Our laboratory has participated in the international workshops on the standardization of the ICA assay, in which its sensitivity was 100%, its specificity was 98%, its validity was 98%, and its consistency was 98% in the most recent round.
IA assay
Insulin antibodies were quantified with a microassay modified from that described by Williams et al. (21). Antibody-antigen complexes were precipitated with protein A-Sepharose (Pharmacia Biotech, Uppsala, Sweden) after incubation of the serum sample for 72 h with mono-[125I]TyrA14-human insulin (Amersham International, Aylesbury, UK) in the presence or absence of an excess of unlabeled insulin. After thorough washing, the samples were transferred from the deep well plates to microtitration plates, scintillation liquid was added, and the bound activity was measured with a liquid scintillation counter (1450 Microbeta Trilux, Perkin-Elmer Corp., Wallac, Inc., Turku, Finland). The specific binding was expressed in relative units (RU) based on a standard curve run on each plate using the MultiCalc software program (Perkin-Elmer Corp., Wallac, Inc.). The cut-off limit for IA positivity was 1.56 RU (99th percentile in 371 nondiabetic Finnish subjects). The disease sensitivity of our microassay was 35% and the specificity 100% based on 140 samples derived from the 1995 Multiple Autoantibody Workshop (22).
GADA assay
Antibodies to the 65 kDa isoform of glutamic acid decarboxylase (GADA) were measured with a radioligand assay as described earlier (23, 24). The results were expressed in RU based on a standard curve run on each plate using a commercial software program (MultiCalc, Perkin Elmer Corp. Wallac, Inc.). The cut-off limit for antibody positivity was set at the 99th percentile in 373 nondiabetic children and adolescents, i.e. 5.35 RU. This assay had a disease sensitivity of 69% and a specificity of 100% based on 140 samples included in the 1995 Multiple Autoantibody Workshop (22).
IA-2A assay
Antibodies to the protein tyrosine phosphatase related IA-2 molecule (IA-2A) were analyzed with a radiobinding assay as described in detail elsewhere (25). The results were expressed in RU based on a standard curve, as for GADA. The limit for IA-2A positivity (0.43 RU) was set at the 99th percentile in 374 nondiabetic Finnish children and adolescents. This assay had a disease sensitivity of 62% and a specificity of 97% based on 140 samples included in the 1995 Multiple Autoantibody Workshop (22).
Data processing and statistical analyses
Elimination time was estimated in months and represents the
mid-age between the last positive and the first negative sample. For
comparison of the elimination dynamics of different uneliminated
antibodies, the proportions of antibodies in samples taken at various
time points were compared with the initial level in the cord blood
sample. The data were evaluated statistically using SPSS software
(SPSS, Inc., Chicago, IL) by means of cross-tabulation and
2 statistics or Fisher’s exact probability
test, the unpaired Student’s t test in the case of normally
distributed variables, and the Mann-Whitney U test and Wilcoxon test
for paired samples in the case of an unequal distribution. Correlation
analyses were performed with the Spearman rank correlation test
(rs).
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Results |
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Twenty-two of the 47 newborn infants (47%) tested positive for ICA in their cord blood, with levels ranging from 5–130 JDF units (median, 8). Thirty-two (68%) were positive for IA, with levels ranging from 1.6–281 RU (median, 9.2); 20 (43%) were positive for GADA, with levels ranging from 5.6–302 RU (median 14.2); and 17 (36%) tested positive for IA-2A, with levels ranging from 0.49–89 RU (median, 4.6). Twenty-three (49%) were positive for only a single antibody, 10 (21%) were positive for 2 antibodies, 8 (17%) were positive for 3 antibodies, and 6 (13%) were positive for all 4 antibodies analyzed.
Correlation with maternal antibodies
The mothers of all 22 ICA-positive infants had ICA, and the mothers of all 32 IA-positive infants had IA in their blood samples taken at the time of the delivery, with ICA levels ranging from 5–130 JDF units (median, 10) and IA levels ranging from 2–600 RU (median, 14). Sixteen of the 20 mothers of GADA-positive infants were GADA positive at the time of delivery, with levels ranging from 5.9–206 RU (median, 37). All 4 mothers who were GADA negative had type 1 diabetes. Three of them had a blood sample taken at the end of the first trimester of pregnancy, and GADA was detectable in that sample (10, 21, and 14 RU). Sixteen of the 17 mothers of IA-2A-positive infants had IA-2A, with levels ranging from 0.43–59 RU (median, 3.4). The mother who was IA-2A negative at the time of delivery had an IA-2A-positive sample at the end of the first trimester of pregnancy (0.63 RU). The antibody levels in the cord blood correlated closely with those in the maternal circulation (ICA: rs = 0.83; P < 0.001; IA: rs = 0.83; P < 0.001; GADA: rs = 0.92; P < 0.001; IA-2A: rs = 0.94; P < 0.001).
Elimination of antibodies and elimination time
One of the infants did not become negative for ICA and IA at all;
thus, his case was excluded from the elimination analysis and will be
discussed in detail later. The prevalences of ICA positivity at the
ages of 3, 6, 9, and 12 months were 33%, 16%, 5.6%, and 0%,
respectively (Table 1
), and those of IA
positivity were 42% at 3 months and 10% at 6 months, whereas no IA
were detected in the samples taken at 9 months. For GADA, 53% tested
positive at 3 months, 22% at 6 months, 17% at 9 months, and 5.9% at
12 months. No maternally acquired GADA was detected at the age of 18
months. For IA-2A the antibody frequencies were 69% at 3 months and
20% at 6 months, whereas no IA-2A could be detected in the samples
taken at 9 months. (Fig. 1) The mean
elimination time for ICA was 3.1 ± 2.7 months (range, 1.5–10.5),
that for IA was 3.1 ± 2.0 months (range, 1.5–7.5), that for GADA
was 4.5 ± 3.9 months (range, 1.5–15.0), and that for IA-2A was
4.3 ± 2.4 months (range, 1.5–9.0). There was no statistical
difference in mean elimination time between the antibodies measured.
Neither the sex of the infant nor the intervention allocation affected
the elimination time. The initial levels of IA, GADA, and IA-2A in the
cord blood correlated closely with the elimination time
(rs = 0.84; P < 0.001,
rs = 0.91; P < 0.001, and
rs = 0.85; P < 0.001,
respectively), whereas there was no significant correlation between the
initial level of ICA and the elimination time (rs
= 0.33; P = 0.15). The mean proportions of the four
antibodies detectable at various time points are shown in Fig. 2.
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The mother in this case had type 1 diabetes and tested positive
for three antibody specificities at delivery (ICA, 8 JDF units; IA, 83
RU; IA-2A, 1.41 RU), and she had also tested positive for GADA in a
sample taken at the end of the first trimester of pregnancy (21.3 RU),
but the sample taken at the time of delivery was just below the cut-off
level (5.2 RU). All four antibodies could be detected in the cord blood
sample (ICA, 5 JDF units; IA, 85 RU; GADA, 10.7 RU; IA-2A, 4.6 RU). The
antibody levels declined until the age of 6 months, but ICA and IAA had
increased again at 9 months. The GADA and IA-2A levels had also
increased by the age of 12 months, and the boy subsequently presented
with clinical type 1 diabetes at the age of 13 months (Fig. 3).
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The estimated mean elimination times for ICA, IA, GADA, and IA-2A varied from 3.1–4.5 months, but considerable variation was seen in the individual times, ranging from 1.5–15 months. Most of the antibodies disappeared from the circulation of an infant within the first 3–6 months, but ICA in 1 and GADA in 3 cases could be detected for a longer time, which confirms the results of our previous study with a smaller number of subjects (16). The higher the initial antibody level, the longer was the elimination time, and the initial antibody level in the cord blood correlated closely with the elimination time for IA, GADA, and IA-2A, but not for ICA. The poor correlation for ICA is probably due to the methodology of the ICA assay, providing semiquantitative antibody levels based on a dilution series of positive samples. No actual studies of the elimination of transplacentally acquired antibodies associated with type 1 diabetes have been performed, but Ziegler et al. (4) found ICA and IA to be very common in the cord blood of infants of mothers with type 1 diabetes, with an ICA prevalence of 21% and an IA frequency of 76%. None of the infants tested positive for ICA in the next sample, taken at the age of 9 months, and less than 5% remained positive for IA. Their more recent analysis of a larger study population suggests that maternal IA can be detected up to the age of 6 months and that signals after that age are likely to be due to de novo synthesis of IAA (27). No maternally acquired IA or IA-2A was detectable after the age of 6 months in our population, but ICA positivity was still seen in 1 infant at the age of 9 months, and GADA remained detectable in another up to the age of 12 months.
The profile of the decline in antibody levels was similar, even though the mean proportion of antibodies still detectable varied from 18–30% at the age of 3 months, from 2.2–14% at 6 months, and from 0.3–15% at 9 months. The higher proportion of uneliminated IA (15%) than of ICA, GADA, or IA-2A at the age of 9 months is probably a consequence of the fact that the initial levels of IA in the cord blood were closer to the cut-off and detection limits, so that the uneliminated proportion will remain higher in subsequent samples. In general, most of the transplacentally transferred antibodies disappear from the circulation of the infant during the first months of life, so that only 20–30% of the antibodies are still detectable at the age of 3 months, about 10% at 6 months, and just a few percent at 9 months. Most of the type 1 diabetes-associated antibodies are of the IgG class (12, 13), and the profile of the decline during the first months of life is similar to the disappearance of maternally acquired IgG antibodies to various virus infections. After birth, the IgG levels decrease rapidly, such that about 30% of IgG is lost during the first week and then the elimination rate slows down; the nadir, 25% of the adult concentrations, is observed at 3–5 months of age, after which the levels start to increase due to endogenous antibody production (28, 29, 30, 31).
The infant who differed from the rest of the group was a boy whose
mother had type 1 diabetes. He tested positive for all four antibodies
measured in his cord blood sample. The levels then declined during the
first months; the lowest levels of ICA, IA, and IA-2A were seen at the
age of 6 months, and the lowest levels of GADA were seen at 9 months.
Thereafter, an increase was observed in all antibody levels as a sign
of de novo production of autoantibodies. Accordingly, the
infant did not become negative for either ICA or IA before the
initiation of endogenous ß-cell autoimmunity leading to clinical
disease by the age of 13 months. This indicates that rising
autoantibody titers in infancy reflect de novo antibody
synthesis, as no other infant showed an increase in his antibody levels
after birth, as illustrated in Fig. 1
.
In summary, we conclude that the antibodies in cord blood represent maternal antibodies and will only rarely be markers of fetal induction of ß-cell autoimmunity. These maternally acquired type 1 diabetes-associated antibodies are in most cases eliminated from the infant’s circulation by the age of 9 months, but may occasionally be seen up to the age of 12 months. Accordingly, when we wish to identify high risk infants for secondary prevention purposes by antibody screening as early as possible, to be able to initiate early preventive measures, we recommend that screening should start at the age of 1 yr. If sampling takes place before that age, a positive result should be confirmed by repeated sampling. The optimal age for the initiation of large scale screening in the general population would be 18–24 months, particularly as only a small proportion of future patients progress to clinical disease before that age.
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Acknowledgments |
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Footnotes |
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Received March 13, 2000.
Revised July 20, 2000.
Accepted July 24, 2000.
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) at 3
months was 18% (median, 0%; range, 0–100%), that of IA (
) was
21% (16%; 1.1–77%), that of GADA (•) was 30% (18%; 5.5–90%),
and that of IA-2A (
) was 20% (18%; 7.2–51%). The corresponding
proportions (median; range) at 6 months were 2.2% (0%; 0–33%) for
ICA, 14% (11%; 0.9–64%) for IA, 10% (1.9%; 0.7–68%) for GADA,
and 6.3% (4.7%; 1.7–16%) for IA-2A; those at 9 months were 0.3%
(0%; 0–4.6%) for ICA, 15.3% (10; 0.4–64%) for IA, 2.3% (1.2%;
0.1–10%) for GADA, and 5.1% (2.7%; 0.6–16%) for IA-2A.