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Original Studies |
Department of Internal Medicine and Endocrine and Metabolic Sciences, University of Perugia, Perugia, Italy
Address correspondence and requests for reprints to: Alberto Falorni, M.D., Ph.D., Department of Internal Medicine and Endocrine & Metabolic Sciences, Via E. Dal Pozzo, 06126 Perugia, Italy. E-mail: falorni{at}dimisem.med.unipg.it
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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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While the prevalence of insulin autoantibodies (IAA) and of IA-2 autoantibodies (IA-2Ab) shows an inverse correlation with the age at onset of type 1 diabetes (9, 10), GAD65 autoantibodies (GAD65Ab) are frequently found in both diabetic children and adults, and the presence of this marker has a high diagnostic sensitivity in the 20–39-yr age group (11).
The presence of GAD65Ab in subjects diagnosed as having type 2 diabetes mellitus identifies a subgroup at high risk for insulin dependency (12, 13). In Japan, the secondary failure to respond to sulphonylurea treatment is retrospectively classified as slowly progressive type 1 diabetes (14, 15), while in western countries the presence of GAD65Ab identifies the so-called latent autoimmune diabetes of the adult (LADA) (16, 17, 18, 19, 20, 21).
Because of the high diagnostic sensitivity for autoimmune diabetes, the presence of GAD65Ab is currently used to identify subjects at high risk for the disease (22, 23). However, the not infrequent occurrence of this marker in healthy subjects and in patients with other autoimmune diseases not necessarily associated with type 1 diabetes, such as the rare neurological stiff-man-syndrome (24), the autoimmune polyendocrine syndrome type I (25), or Graves’ disease (26), limits its diagnostic specificity. In adult diabetic subjects, the concomitant presence of islet cell antibodies (ICA) improves the predictive value of GAD65Ab for insulin requirement (19).
The analysis of the variable region of human monoclonal autoantibodies revealed that the production of diabetes-associated GAD65Ab is the result of an autoantigen-driven process, which selects high-affinity autoantibodies (27). During the natural history of childhood type 1 diabetes, autoantibodies directed to highly conserved epitopes located in the 240–360 (middle: M) and 451–570 (carboxyterminal: C) amino acid regions of the autoantigen (28, 29, 30, 31, 32) are selected. Production of epitope-specific GAD65Ab may require a permissive genetic background as GAD65Ab found in stiff-man-syndrome or in the autoimmune polyendocrine syndrome type I, both of which have different genetic associations than type 1 diabetes, are directed to a different and broader spectrum of epitopes (31, 33, 34). Thus, the mechanisms of GAD65Ab production may be different in different diseases. Preliminary data suggest that an increase in GAD65Ab to COOH-terminal epitopes distinguishes diabetic from healthy children (32). Although it has been proposed that LADA-associated GAD65Ab would recognize both a middle and a carboxyterminal epitope (35), it is still unclear what is the diagnostic sensitivity and specificity of GAD65Ab epitopes for insulin requirement in adult diabetic subjects with slowly progressive type 1 diabetes. During long-term chronic autoimmunity, either spreading or restriction of autoantibody epitopes may occur. Because LADA is the result of a long-term, chronic autoimmune process, the accurate localization of GAD65Ab epitopes associated with this form of diabetes would provide important information to our understanding of the molecular mechanisms of diabetes-associated autoantibody production. In addition, the identification of epitope-specific GAD65Ab with high diagnostic specificity for insulin requirement may be instrumental in the accurate diagnosis of LADA subjects.
The major aim of our study was to estimate the diagnostic sensitivity and specificity for insulin requirement of GAD65Ab epitopes in subjects with adult-onset diabetes. We took advantage of cDNAs for chimeric molecules generated by substitution of regions of human GAD65 with homologous regions of GAD67, which have been previously used to estimate the diagnostic accuracy of GAD65Ab epitopes for childhood type 1 diabetes (32). We first identified a group of GAD65Ab-positive subjects from a large population of adult patients diagnosed with and treated for type 2 diabetes mellitus. We then tested the diagnostic sensitivity and specificity of epitopes of GAD65Ab for insulin requirement. Finally, the risk for thyroid autoimmunity, as made evident by the presence of thyroid peroxidase (TPO) autoantibodies, was evaluated in relation to the occurrence of epitope-specific GAD65Ab. Although our initial screening of type 2 diabetic subjects was cross-sectional, follow-up samples from some of the GAD65Ab-positive subjects were available and were used to study the time-related modifications of levels of GAD65Ab epitopes.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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A total of 569 Italian adult diabetic subjects were studied in
the initial screening for GAD65Ab. Subjects were randomly selected
among those consecutively attending the diabetes unit of our department
between October 1997 and March 1999. Inclusion criteria were: 1) an age
of 25 yr or more at the time of the clinical diagnosis of diabetes
mellitus, 2) classification as Type 2, noninsulin-dependent diabetes
mellitus, and 3) treatment with diet and/or hypoglycemic agents for at
least 1 yr after the initial diagnosis. The clinical features of the
patients enrolled in our study are summarized in Table 1
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An informed consent was obtained from all the study participants.
GAD65Ab assays
GAD65Ab were determined using a radiobinding assay with in vitro translated recombinant human 35S-GAD65 (36, 37). The cDNA for human GAD65 (38, 39) was a kind gift of dr. Å Lernmark (University of Washington, Seattle, WA). GAD65Ab levels were expressed as a relative index (GAD65 index), using one positive standard serum from a type 1 diabetic subject and two negative control sera from healthy subjects, in each assay. Our autoantibody assay was validated by participation in international workshops for the standardization of islet autoantibody determinations (40, 41, 42), and the upper level of normal was 0.035, as calculated as the mean + 3 SD of the GAD65 index observed in healthy controls. Using this cut-off value, the diagnostic sensitivity and specificity measures of our GAD65Ab assay, as evaluated in the 1995 Combined Autoantibody Workshop (lab code AT) (42), were 85% and 100%, respectively.
To localize the GAD65Ab epitopes associated with LADA, we used radiobinding antibody assays with in vitro translated 35S-GAD65/GAD67 chimeric molecules, generated by substituting GAD65 regions with homologous regions of rat GAD67 (32). The cDNA for rat GAD67 (43) was a kind gift of dr. B. Michelsen (Hagedorn Research Institute, Gentofte, Denmark). The DNA coding for a chimeric molecule containing the NH2-terminal region of rat GAD65 (aa. 1–83) in fusion with the central-COOH terminal regions of rat GAD67 (aa. 89–593) (44) (GAD65-N-67) was a kind gift of dr. P. De Camilli (Yale University, New Haven, CT). GAD65-M was generated by substituting the amino acid region 361–585 of human GAD65 with the region 369–593 of rat GAD67 (GAD651–360/GAD67369–593), and it contained the middle epitopes of GAD65Ab; GAD65-C was generated by substituting the amino acid region 1–436 of human GAD65 with the region 1–445 of rat GAD67 (GAD671–445/GAD65437–585) and contained the COOH-terminal epitopes of GAD65Ab (32). The rabbit antiserum R7309 (45), specific for the region 4–22 amino acids of human GAD65, was used as positive standard serum in the GAD65-M and GAD65-N-67 antibody assays, and the rabbit antiserum R10266 (45), specific for the region 2–19 amino acids of rat GAD67, was used as positive standard serum in the GAD67 and GAD65-C antibody assays. Epitope-specific GAD65Ab levels were expressed as relative indices (GAD67 index, GAD65-N-67 index, GAD65-M index, and GAD65-C index) using 2 negative control sera from healthy individuals. Using the mean + 3 SD of the results obtained with the sera from 100 healthy subjects, the upper level of normal of the GAD67Ab assay was 0.02, that of the GAD65-N-67Ab assay was 0.02, that of the GAD65-MAb assay was 0.02, and that of the GAD65-CAb assay was 0.025. Sera from all the GAD65Ab-positive adult diabetic subjects identified in our study were separately tested in all the antibody assays using GAD67 or the three forms of GAD65/GAD67 chimeric molecules. To correct for the inter-assay variation, all the follow-up samples were analyzed in a single assay. The intra-assay coefficients of variation were 5% for the GAD65Ab assay, 7% for the GAD67Ab assay and for the GAD65-N-67Ab assays, 6% for the GAD65-MAb assay, and 8% for the GAD65-CAb assay.
Thyroid peroxidase antibody assay
Thyroid peroxidase (TPO) autoantibodies (TPOAb) were determined using an immunoradiometric assay with recombinant human 125I-TPO (Radim, Angleur, Belgium). Results were expressed as international units per milliliter of bound TPO, and the upper level of normal was 130 IU/mL.
C-peptide assay
Basal plasma C-peptide values were evaluated using a commercially available RIA kit (Diagnostics Systems Laboratories, Inc., Webster, TX). Sensitivity of the assay was 0.003 nmol/L.
Statistical analysis
Differences in frequency of autoantibodies were tested by
the 
-square test with Yates’ correction, whenever appropriate, or
by the Fisher’s exact test. Differences in median and range were
tested by the nonparametric Mann-Whitney test. Differences in
autoantibody levels during the follow-up were tested by the
nonparametric Wilcoxon test for paired samples. Autoantibody levels
were not normally distributed in diabetic subjects and were therefore
logarithmically transformed. The relationship between autoantibody
levels and the duration of diabetes was analyzed by linear regression.
The dependence of the dichotomous GAD65Ab positivity on several other
variables–age, gender, disease duration, type of therapy, BMI and
basal C-peptide–was assessed by logistic regression analysis. A
p value less than 0.05 was considered significant. All
statistical tests were performed by the Statistical Package for Social
Sciences for Windows for personal computers (SPSS, Inc.,
Chicago, IL, USA).
Sensitivity and specificity of GAD65Ab and GAD65Ab epitopes for insulin requirement were percentage of antibody-positive subjects among insulin treated patients (true positives) and percentage of antibody-negative subjects among noninsulin treated patients (true negatives), respectively.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Using in vitro translated human autoantigen, GAD65Ab were found in 61/569 (10.7%) Italian subjects with an initial diagnosis of type 2 diabetes mellitus. A total of 50/61 (82%) GAD65Ab-positive subjects had already been converted to a treatment with insulin at the time of the study.
Using logistic regression analysis, the presence of GAD65Ab in adult
diabetic subjects depended on (in order of decreasing strength) insulin
therapy (P < 0.0001), low BMI (P <
0.0001), and low basal C-peptide values (P = 0.01)
(Table 1
). When the prevalence of GAD65Ab was evaluated in relation to
the type of therapy, GAD65Ab were found in 50/217 (23.0%) subjects in
treatment with insulin and in only 11/352 (3.1%) subjects in treatment
with oral hypoglycemic agents and/or diet.
In autoantibody-positive subjects, GAD65Ab levels were significantly
higher in insulin-treated subjects (GAD65 index median: 0.992; range:
0.12–3.02) than in noninsulin treated ones (GAD65 index median: 0.258;
range: 0.066–1.45) (Fig. 1)
(P = 0.002). Furthermore, GAD65Ab levels correlated
positively with the duration of diabetes (r = 0.29,
P = 0.025). In the presence of GAD65Ab, no statistical
significant difference in disease duration (as estimated in years after
the initial diagnosis of type 2 diabetes mellitus) was observed between
the insulin-treated (median: 8 yr, range: 1–29 yr) and the
noninsulin-treated (median: 6 yr, range: 1–46 yr) patients.
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GAD65Ab epitopes in LADA
Based on the results of the initial screening for the presence of GAD65Ab, we subsequently tested the diagnostic sensitivity and specificity of epitopes of GAD65Ab for insulin requirement.
A total of 20/61 (32.8%) GAD65Ab-positive subjects had a GAD67 index above the upper level of normal. The presence of antibodies reacting with GAD67 was associated with high levels of GAD65Ab (GAD65 index median: 1.42, range: 0.124–2.59). Antibodies to the GAD65-N-67 chimera were found in 16/61 (26.2%) GAD65Ab-positive subjects. A total of 15 of the 16 samples found positive in the assay using the GAD65-N-67 chimera were found positive also for GAD67Ab.
Because the majority of GAD65Ab-positive samples were negative for both GAD67Ab and GAD65-N-67Ab and because the presence of GAD65-N-67Ab was strongly associated with that of GAD67Ab, we concluded that the GAD65Ab detected in our LADA patients was primarily directed to epitopes located in the middle and COOH-terminal regions of GAD65.
A total of 48/61 (78.7%) and 60/61 (98.4%) GAD65Ab-positive adult diabetic subjects demonstrated positive results in the assays using GAD65-C or GAD65-M chimeras, respectively. The simultaneous presence of GAD65-M and GAD65-C autoantibodies was demonstrated in 47/61 (77%) cases. All of the 20 samples found positive for GAD67Ab, as well as all of the 16 samples found positive for GAD65-N-67Ab, were also positive for both GAD65-MAb and GAD65-CAb. We found GAD65-MAb in 13 subjects (21.3%) negative for GAD65-CAb. Conversely, GAD65-CAb were present in only 1 subject (1.6%) in the absence of GAD65-MAb.
We then analyzed the presence of epitope-specific GAD65Ab in relation
to the type of therapy at the time of the study (Fig. 1 and Table 2). Out of the 50 GAD65Ab-positive
subjects in treatment with exogenous insulin, 45 (90%) were positive
for both GAD65-MAb and GAD65-CAb; 1 (2%) was positive only for
GAD65-CAb; and 4 (8%) were positive only for GAD65-MAb. Out of the 11
subjects in treatment with hypoglycemic agents and/or diet, 2 (18.2%)
were positive for both GAD65-M- and GAD65-CAb, and 9 (81.8%) were
positive only for GAD65-MAb in the absence of GAD65-CAb. The frequency
of GAD65-CAb was significantly higher in insulin-treated subjects
(46/50, 92%) than in noninsulin treated (2/11, 18.2%) subjects
(P < 0.0001). Conversely, the presence of GAD65-MAb in
the absence of GAD65-CAb was significantly lower in insulin-treated
(4/50, 8%) than in noninsulin-treated (9/11, 81.8%) subjects
(P < 0.0001). Furthermore, the clinical features of
subjects positive for GAD65-MAb only (Table 1) were indistinguishable
from those of GAD65Ab-negative individuals. On the contrary, the
presence of autoantibodies directed to both the middle and the
carboxyterminal epitope regions identified a subgroup of patients with
low BMI and need for insulin requirement (Table 1). Basal C-peptide
values were significantly lower in the subjects positive for both
GAD65-M and -CAb (median 0.023 nmol/L, range 0–0.67) than in the
subjects positive for GAD65-MAb only (median 0.46 nmol/L, range 0–2.0)
(P = 0.01) (Table 1).
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and correlated with those of GAD65Ab (r2 = 0.74,
P < 0.001 and r2 = 0.76,
P < 0.001, respectively). In addition, levels of
GAD65-MAb and GAD65-CAb correlated with each other
(r2 = 0.71, P < 0.001).
Overall, the samples positive for both GAD65-M and GAD65-CAb had
GAD65Ab levels (GAD65 index = median:1.102, range:0.12–3.021)
higher than those observed in the samples positive for GAD65-MAb only
(GAD65 index = median:0.342, range:0.066–0.587)
(P < 0.01). Furthermore, the two samples from
noninsulin-treated subjects found positive for both GAD65-M and
GAD65-CAb were those with the highest GAD65 index (1.357–1.445) (Fig. 1). However, when samples with a GAD65 index equal to or lower than
0.587 (the highest GAD65 index associated with the presence of
GAD65-MAb only) were taken into consideration, 11/15 (73.3%)
insulin-treated subjects and 0/9 noninsulin-treated subjects were
positive for GAD65-CAb (P = 0.0006).
The sensitivity of islet autoantibodies for insulin requirement ranged
from 1.8% of GAD65-MAb only to 23% of GAD65Ab (Table 3). The specificity of GAD65-CAb (99.4%)
was higher than that of either GAD65Ab or GAD65-MAb only (Table 3).
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Figure 2 shows the levels of
GAD65Ab, GAD65-MAb, and GAD65-CAb in 16 insulin-treated subjects during
follow-up. In 12/16 subjects, the levels of GAD65Ab increased during
the follow-up period (P = 0.045) (Fig. 2, panel A). In
addition, the levels of GAD65-CAb increased during the follow-up period
in 14/16 subjects (P < 0.01) (Fig. 2, panel B). On the
contrary, levels of GAD65-MAb did not increase significantly at
follow-up (Fig. 2, panel C).
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TPO antibodies in LADA
The presence of TPOAb was evaluated in 61 GAD65Ab-positive
subjects and in a group of 174 diabetic subjects (92 males and 82
females) of similar age, but negative for GAD65Ab. TPOAb occurred more
frequently in GAD65Ab-positive (15/61, 24.6%) (median levels: 555
UI/mL, range: 162-1766) than in GAD65Ab-negative (9/174, 5.2%) (median
levels: 909 UI/mL, range: 144-2556) subjects (P <
0.0001) (Table 1). In the presence of GAD65Ab, 14/15 (93.3%)
TPOAb-positive subjects were in treatment with insulin at the time of
our study. Similarly, 14/15 (93.3%) TPOAb-positive subjects had
antibodies directed to both GAD65-M and GAD65-C. Accordingly, TPOAb
were present in 14/47 (29.8%) subjects positive for both GAD65-M and
C-Ab (P < 0.0001 vs. GAD65Ab-negative
subjects), but in only 1/13 (7.7%) with GAD65-MAb only.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In our study, we addressed the problem of the selection of GAD65Ab epitopes in adult subjects initially diagnosed with and treated for type 2 diabetes mellitus. We observed that GAD65Ab was present in 10.7% of the studied population from central Italy and that the presence of autoantibodies binding to the COOH-terminal region of GAD65 was strongly associated with the need for insulin therapy.
The relatively high frequency of GAD65Ab in type 2 diabetic subjects is highly relevant for the understanding of the pathogenesis of autoimmune diabetes and for the classification of adult-onset diabetes mellitus. It must be noted that our study was carried out at a WHO collaborating center for the improvement of quality of diabetes care and that our GAD65Ab assay had a very high diagnostic sensitivity and specificity. Accordingly, it is unlikely that the results of our study were influenced by an inaccurate clinical diagnosis at the time of the first visit or by an inadequate accuracy of our assay. Rather, our GAD65Ab frequency of 10.7%, similar to those recorded in several other studies, confirms that slowly progressive type 1 diabetes mellitus has been so far underdiagnosed. On the other hand, it must be noted that 38% of our type 2 diabetic subjects were in insulin treatment at the time of our study. Thus, the majority of insulin-treated subjects was GAD65Ab-negative.
The positive correlation between GAD65Ab levels and disease duration supports the hypothesis that a long-term, chronic, autoantigen-specific process is sustained for several years in adult subjects with latent and slowly progressive autoimmune diabetes. This conclusion is also supported by our data on follow-up samples. Thus, GAD65Ab can be considered a sensitive marker of chronic islet autoimmunity. However, the mechanisms responsible for maintaining a GAD65-specific autoimmune process with slowly progressive ß-cell destruction have not yet been clarified.
The presence of GAD67Ab in type 1 diabetic subjects is associated with high levels of GAD65Ab and seems to be the result of a cross-reactivity of the two isoforms of the enzyme with some GAD65Ab (32, 36). Thus, chimeric GAD65/GAD67 molecules can be used in epitope-specific assays. This strategy has previously proven successful in localizing conformation-dependent GAD65Ab epitopes (29, 30, 31, 32). The strong association between GAD65-N-67Ab and GAD67Ab observed in our study suggests that LADA-associated GAD65Ab is not directed to the N-terminal end of the autoantigen. This is in line with similar data obtained when using samples from type 1 diabetic children (32). Our finding of a high association between the appearance of COOH-terminal epitope-specific GAD65Ab and insulin requirement, with a specificity as high as 99.4%, can be interpreted to indicate that the selective production of epitope-specific autoantibodies marks the need for insulin treatment.
A previous study (32) showed that GAD65Ab binding to both the middle and the COOH-terminal region of the autoantigen is present in more than 80% of antibody-positive type 1 diabetic children. Furthermore, a selective increase in COOH-terminal specific autoantibodies distinguished diabetic from healthy children (32). Our data on LADA subjects show that the concomitant presence of GAD65-M and GAD65-C Ab (GAD65-M+CAb) is a feature also of adult-onset autoimmune diabetes. In addition, detection of GAD65-M + C Ab in samples collected years before the development of insulin dependency supports the hypothesis that this immune marker can predict insulin requirement.
GAD65-MAb only has been found in 7% of type 1 diabetic children (32), a frequency similar to that now documented in LADA subjects in insulin therapy (8%). In addition, we identified a group of subjects positive only for GAD65-MAb in treatment with oral hypoglycemic agents and/or diet. At this stage, it is not yet clear whether middle antibody epitopes associated with childhood diabetes are identical to those observed in adult patients. Nevertheless, our data are consistent with the existence of a subgroup of GAD65Ab-positive adult-onset diabetic subjects with phenotypic characteristics similar to those of the typical type 2, GAD65Ab-negative, subjects. This subgroup is identified by the exclusive presence of GAD65Ab binding to the middle region of the autoantigen (GAD65-MAb only).
Several hypotheses can be formulated to explain the existence of two subgroups of GAD65Ab-positive subjects. The presence of GAD65-MAb, in the absence of GAD65-CAb, might be the result of a different sensitivity of the two epitope assays. However, it must be noted that, in the previous study on 155 type 1 diabetic children (32) that used the same chimeric molecules, no difference in diagnostic sensitivity was observed between the two epitope assays. In our present study, the presence of GAD65-MAb only could not be explained simply by a low GAD65 index and by a higher sensitivity of this assay, compared with that for GAD65-CAb. In fact, when we analyzed adult subjects with low levels of GAD65Ab (to standardize for the sensitivity of the GAD65-MAb assay), the presence of GAD65-CAb could be detected only in insulin-treated subjects but not in patients in treatment with oral hypoglycemic agents and/or diet. Thus, the observed differences in GAD65Ab epitope selection are more likely due to a biological phenomenon than to a methodological artifact.
At the present stage, we cannot rule out the possibility that the selective production of GAD65-MAb represents an early phase of the GAD65-specific autoimmune process. Future prospective studies will address this specific question. However, several lines of evidence tend to favor the hypothesis that GAD65-MAb identifies a distinct subgroup of patients with low risk for insulin requirement. Firstly, disease duration was similar in the two subgroups of GAD65Ab-positive subjects (GAD65-MAb only and GAD65-M+CAb). Second, we did not observe any switch in epitope specificity in our limited follow-up samples. None of the 5 subjects positive for GAD65-MAb only, of whom follow-up serum samples were available, became positive for GAD65-CAb during a follow-up period of 10–21 months. Third, and more important, we observed the exclusive presence of GAD65-MAb in two subjects still in treatment with oral hypoglycemic agents after 22 yr and 46 yr of disease duration, respectively. Finally, the prevalence of thyroid autoantibodies was significantly increased only in patients positive for GAD65 M+CAb, but not in those positive for GAD65-MAb only, as compared to GAD65Ab-negative subjects. This supports the hypothesis that only patients positive for GAD65-M+CAb have an increased risk for endocrine autoimmunity. If the selective presence of GAD65-MAb only is not directly related to an islet autoimmune process, it may be the result of a cross-reactivity with other, unrelated antigens.
In conclusion, our study demonstrates that, in adult subjects diagnosed with type 2 diabetes, the presence of GAD65Ab is associated with characteristics typical of a slowly progressive type 1 diabetes with high probability of need of exogenous insulin. Based on epitope specificity, two subgroups of subjects with GAD65Ab have been identified with different phenotypic characteristics and different risk for insulin requirement. The use of epitope-specific assays can improve the diagnostic specificity of GAD65Ab for insulin requirement.
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Acknowledgments |
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Footnotes |
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Received June 29, 1999.
Revised August 30, 1999.
Accepted September 3, 1999.
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 A. FALORNI and F. CALCINARO Autoantibody Profile and Epitope Mapping in Latent Autoimmune Diabetes in Adults Ann. N.Y. Acad. Sci., April 1, 2002; 958(1): 99 - 106. [Abstract] [Full Text] [PDF]
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A. COSENTINO, G. GAMBELUNGHE, C. TORTOIOLI, and A. FALORNI CTLA-4 Gene Polymorphism Contributes to the Genetic Risk for Latent Autoimmune Diabetes in Adults Ann. N.Y. Acad. Sci., April 1, 2002; 958(1): 337 - 340. [Abstract] [Full Text] [PDF]
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G. Gambelunghe, M. Ghaderi, C. Tortoioli, A. Falorni, F. Santeusanio, P. Brunetti, C. B. Sanjeevi, and A. Falorni Two Distinct MICA Gene Markers Discriminate Major Autoimmune Diabetes Types J. Clin. Endocrinol. Metab., August 1, 2001; 86(8): 3754 - 3760. [Abstract] [Full Text] [PDF]
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 D. B. Sacks and A. Lernmark Molecular Manipulation of Autoantibody Testing in Type 1 Diabetes: Two for One Clin. Chem., May 1, 2001; 47(5): 803 - 804. [Full Text] [PDF]
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