Acta Paediatr 01: 593-7. 1992
Diabetes-related autoantibodies do appear in children with coeliac disease U Di Mario', E Anastasi', P Mariani2, G Ballati2, R Perfetti', P Triglione2, M Morellini3 and M Bonamico2 Department of Endocrinology', Department of Paediatric? and Department of Experimental Medicine', University "La Sapienza". Rome, Italy
Di Mario U, Anastasi E, Mariani P, Ballati G, Perfetti R, Triglione P, Morellini M, Bonamico M. Diabetes-related autoantibodies d o appear in children with coeliac disease. Acta Paediatr 1992;81: 593-7. Stockholm. ISSN 0803-5253 . Humoral immune factors related to type 1 diabetes have been investigated in children with coeliac disease. Anti-insulin (IAAb), immunoglobulin (aIgAb), islet cell (ICA) and glucagon autoantibodies were examined in 15 children with coeliac disease at diagnosis (group I), in 15 children with coeliac disease following a gluten-free diet (group 2) and in 30 control patients (groups 3 and 4). IAAb were present in 27% of group 1 and in 20% of group 2 patients and aIgAb were significantly increased in group 1 and 2 patients; two patients in group 2 were positive for ICA; none of the coeliac disease patients were positive for anti-glucagon antibodies. The levels of anti-gliadin antibodies in group 1 were positively correlated with those of aIgAb. Coeliac disease-related HLA antigens were not correlated with antibody presence. The presence of diabetes-related humoral immune factors in coeliac disease raises the question as to whether or not they are predictive of subclinical pancreatic damage or whether they are simply indicators of a more general autoimmune diathesis. 0 Anti-immunoglobulin antibodies, coeliac disease, insulin autoantibodies, type I diabetes
U Di Mario. Via Belluno, 8 , 00161 Roma, Italy
The pathogenesis of coeliac disease (CD) is still obscure despite a growing body of evidence suggesting a role for immunological abnormalities in the sequence of causative events (1-5). Type 1 diabetes is one of the organ-specific autoimmune disorders ( 6 ) which has been described as associated with C D (7-9). The link between C D and type I diabetes is indirectly reinforced by genetic studies which have shown that these disorders share an increased correlation with HLA-DR3, whereas other alleles tend to segregate either with C D or diabetes, respectively (10, 11). However, it is still uncertain whether the clinical association between the two disorders is due to the to some extent common genetic background or to the presence of shared pathogenetic mechanisms. In this study we have evaluated those immunological abnormalities in C D patients considered to be highly indicative of diabetes, both during the slowly progressing active stages of C D and during the gluten-free diet remission periods. Consequently, we examined autoantibodies directed towards insulin and autologous immunoglobulins, recently described as being associated with diabetes (1 2, I3), together with anti-islet cell antibodies, anti-glucagon autoantibodies and anti-gliadin antibodies in HLA-typed C D patients both before and during specific diet treatment.
Materials and methods Patients
Thirty children with C D and 30 control patients were included in the study. The diagnosis of CD, was made according to ESPGAN criteria (14). Anti-gliadin antibodies and small bowel mucosa biopsy were used as diagnostic criteria. The C D patients (seven males, 23 females; median age at the time of the study eight years, interquartile range (IR) 11 months-18 years) were grouped according to diet regimen: 15 subjects were on a normal diet (group I), whereas 15 were on a gluten-free diet (group 2) for an average duration of three years (IR = 11 months-four years one month). In addition, 16 children (eight males, eight females; median age five years three months, IR = two years two months-eight years nine months) with irritable bowel syndrome (toddler's diarrhoea and recurrent abdominal pain) (group 3) diagnosed after exclusion of organic pathologies by a series of tests, and 14 hospitalized children in a surgery department (10 males, four females; median age four years six months, IR =two years nine months-six years one month) with no infectious, immunologic, gastrointestinal or other coeliac-related diseases (group 4) were recruited to act as controls.
U Di Mario et ul.
Normal subjects with no history of diabetes or C D were used as controls in order to evaluate the limit of positivity in the different techniques used. Ant i-gliadin antibodies The anti-gliadin (IgA and IgG) antibodies (AGA) were determined using an ELISA method (15). Sera were considered positive when absorbance was higher than normal samples (63 subjects) by 3SD.
Insulin autoantibodies Insulin autoantibodies (IAAb) were assessed using a radioimmune displacement assay; the method used was a modified version of an assay described previously (1 6, 17) and is essentially the method reported by Vardi et al. (18) with minor modifications (19). The precision of the test, calculated as the S D of the duplicate geometric value difference, was 0.014. The reproducibility in intraand inter-assay determinations of three positive and two negative samples was 8.6 and 15.3, respectively. The limit of positivity, calculated as the mean + 3 SD of 81 normal values was 30 pU/l. Anti-immunoglobulin antibodies Anti-immunoglobulin antibodies (aIgAb) were assessed using a radioimmune assay developed in our laboratories (12). The limit of positivity (66 pg/l) of 104 normal values was calculated as the 90th percentile, practically coinciding with the mean + 2 SD. Anti-islet cell antibodies and anti-glucagon antibodies Islet cell antibodies (ICA) and anti glucagon antibodies were tested by indirect immunofluorescence on unfixed cryostat sections of human pancreas. The ICA technique used in our laboratories has demonstrated accuracy and precision as determined by the 3rd Juvenile Diabetes Foundation (JDF) International Workshop on ICA standardization (Grade A Laboratory, regression coefficient for blinded sera: 0.94) (20). Results are expressed in J D F units. Immunoglobulins ‘gA and IgG immunoglobulins were measured by the nephelometric method.
ACTA PRDIATR 8 I ( 1992)
phocytes using a standard National Institute of Health technique; B-lymphocyte enriched preparations were obtained by filtration on nylon fibres as described previously (21, 22). Typing sera were selected from the Ninth International Histocompatibility Workshop and correlated local sera were used (1 1). Statistical evaluations Fisher’s exact test (F), the chi-square test for multiple comparison (X), Wilcoxon’s test (W), Pearson’s coefficient (P) and the Cox test for trends in proportions (C) were used, where appropriate, to evaluate the results.
Results Insulin autoantibodies
IAAb positivity was observed in 23.3% of C D patients (Table 1). Medians (IR) of IAAb values were 13 (5-38), 4 (l-l4), 4 (1-13) and 5 (1-17.5) pU/l in groups 1, 2, 3 and 4, respectively (Fig. 1). There was a significant difference in IAAb levels between groups 1 and 3 or 4 ( p < O . O I ; W, C) and between groups 2 and 3 or 4 (p