NUTRITIONAL PRIMARY PREVENTION OF TYPE 1 DIABETES IN CHILDREN (TRIGR)

An intriguing question is the mechanism by which CMP may cause the lesion of the pancreatic ß-cells. Several mechanisms have been proposed, e.g. molecular mimicry between a fraction of bovine serum albumin (BSA) and a ß-cell antigen, a possible deleterious effect of other whey protein components of CM, such as ß-lactoglobulin, and of ß-casein, bovine insulin, etc (reviewed in ref. 7). A recent ecological study indicated that the diabetogenicity of CM might bedue to the A1 variant of bovine ß-casein (8). Regardless of the mechanism, we consider the indirect evidence from animal experiments and observations in man, particularly from the experience from our second pilot study in Finland, described below under b.a.c., and the risk linked with early supplementation of CMP reviewed briefly above, to be sufficient to justify a dietary intervention trial. The formula to be used, a casein hydrolysate, does not contain whey proteins, such as BSA or ß-lactoglobulin, and the small peptides of the casein component (<1500 D) are characterized by low immunogenicity. This formula has been shown to be non-diabetogenic in the NOD mouse (9). In the second pilot of the present TRIGR-study in Finland the use of the test formula resulted in lower prevalence of diabetes-related autoantibodies in children at increased genetic risk of type 1 diabetes (10).

We certainly agree with the notion that prospective studies, considering both the timing and duration of exposure to CMPs are worthwhile, preferably also in different populations, as the genetic-environmental interactions may vary from one population to another (12). But even prospective studies do not seem to give uniform answers: the question of a possible effect of BF vs. early supplementation with CMP containing formula on the development of autoimmune markers of type 1 diabetes has been addressed by several research groups. The DAISY group in Denver, the German BABY-DIAB Study Group and the Australian Baby DIAB Study Group have reported the absence of an association between the duration of BF or introduction of CM, and the development of islet autoimmunity in children with first degree relatives (FDR) with type 1 diabetes (13-15). However, in the Finnish Diabetes Prediction and Prevention (DIPP) Study the opposite has been observed: the series consists of children with an increased genetic risk, identified from the general population. In a nested case-control study the first 65 children who seroconverted to ICA positivity (cases) before the age of 4 years and 390 ICA negative control subjects (six controls/case), matched for date of birth, sex and HLA-DQB1-genotype, were compared. The risk of IA-2A positivity or positivity for all four antibodies (ICA, IAA, GADA and IA-2A) was lower in children exclusively breast-fed for more than 3 months compared to those who were breast-fed for 3 months or less. Also infants in whom supplementary milk feeding had started after the age of 3 months were at lower risk of IA-2A positivity or positivity for all four autoantibodies (16). The associations remained significant after adjustment for maternal age, duration of maternal education and 12 month relative weight and height of the child. These observations suggest that short BF and early introduction of CM based infant formula predisposes young children at increased genetic risk for diabetes to progressive signs of ß-cell autoimmunity.

There are many indications by now that the pathogenetic process leading to manifest type 1 diabetes may start in some cases very early in life, even in utero (17,18). In addition, during childhood the incidence of the disease has over the last decade shifted towards younger age in several countries (19). Therefore, studies on environmental factors in the first years of life, among them the role of CMPs are crucial. CMPs are unique in one respect: in industrialized countries they are the first foreign proteins entering the infant gut, since most infant formulas are CM based. In a critical review of the hypothesis, published in August 1999, it was stated that the real debate is about mucosal immune function as to the possible role of CM in the pathogenesis of type 1 diabetes (11), which is in line with a proposal from our group some years ago in relation to the role of CMPs in the pathogenesis of type 1 diabetes (20,21).

In the second pilot study 207 children with an increased genetic risk (first degree relative(s) with type 1 diabetes, HLA DQB1*0302 and/or DQB1*02 positive, absence of DQB1*0602, *0603 and *0301) have been followed for the observation period of 2 years. The distribution of the HLA DQB1*02/0302, *0302/x and *02/y genotypes was rather similar in the two study groups. The infants received after exclusive BF in a double blind, randomized design either a casein hydrolysate formula or a conventional CM-based formula until the age of 6-8 months. Antibodies to GAD65, IA-2 and insulin (IAA) were assayed by radiobinding assays, and islet cell antibodies ( ICA ) by immunofluorescence. By 24 months of age, 5/98 (5.1 %) of the subjects in the casein hydrolysate group had developed at least one autoantibody, whereas 14/109 (12.8 %) in the control group had done so (P=0.09, Fisher's exact test). IAA were the first to appear in the latter group. Theeffect of the intervention on the risk of seroconversion of any of the four autoantibodies was as follows: casein hydrolysate vs. CM-based formula, risk ratio (RR) 0.40 (95% CI 0.13-1.03), p=0.059. As there were some differences between the two groups in the duration of BF and formula feeding, the analysis was done by adjusting for the duration of study formula feeding, the RR being 0.34 (0.11- 0.89), p=0.027. These findings are supportive of the hypothesis and justify moving to a full study described in this proposal.

These preliminary findings suggest that elimination of dietary CMPs and replacement with hydrolyzed casein formula over the first 6-8 months may modulate the appearance of diabetes associated autoantibodies in children at increased risk for type 1 diabetes over the first 2 years of life.They also demonstrate for the first time in man that it may be possible to modify spontaneous ß -cell autoimmunity by dietary intervention.

It is our firm opinion that the CMP and type 1 diabetes issue will not be solved without a properly planned, sufficiently powerful, prospective, double-blinded randomised intervention trial. Association studies have an inherent weakness in identifying a ubiquitous, noxious agent, such as CMP. For instance, the cause of coeliac disease would likely not have been discovered were it not for the unavailability of wheat in the Dutch World War II diet (22).The prevailing controversies make an intervention study even more important than earlier.

b.b. Significance

The consequences and costs of type 1 diabetes presenting in childhood are immense, not only economically and for the society, but also with regard to human life. Microvascular complications develop in a considerable proportion of the patients in the course of time affecting their quality of life. Those individuals manifesting type 1 diabetes in childhood and adolescence may have a severalfold increased risk of macrovascular complications in adult life. It is therefore easy to realize benefits which would result if we could prevent type 1 diabetes even in a part of the cases. The economic burden of type 1 diabetes is admittedly difficult to assess, the costs being composed of three elements: direct, indirect and psychological (23). However, it has been assessed that the life-time cost of an individual contracting type 1 diabetes in childhood may reach 1 million USD. Thus it is evident that the high incidence of type 1 diabetes, like in Finland and several other countries participating in this project not only carries a great social but also a heavy economical impact as well. It is of interest to note the recent increasing attention by WHO in early nutrition in the context of the etiopathogenesis of type 1 diabetes (24), and it has been proposed that research should respond with a new focus on early events (25). Our research proposed does just that. A positive outcome of the present proposal would thus have a major input on the economic and social consequences of type 1 diabetes.

The accumulated data suggest the simple and testable hypothesis that temporary but strict avoidance of CMPs in the vulnerable early infancy period could prevent the development of type 1 diabetes in genetically susceptible children. If the hypothesis is proven correct, primary prevention of type 1 diabetes and avoidance of the associated morbidity, mortality and health care costs would for the first time become a realistic goal. An additional benefit is the possibility to gain important insight in to the immunological events in high risk infants during the first years of life, so poorly understood so far, and into the natural course of those infants who have been on the control formula. As about half of the children in our study are control subjects, we can also learn of the natural history of the development of islet cell related antibodies and other measured variables in this part of the study.

We like to emphasize, that this is an innovative trial, being the first ever started primary prevention trial against type 1 diabetes. In contrast to the current secondary prevention strategies which involve extensive genetic and/or autoimmune marker screening, the innocuous present strategy could be directly applied to the general population from where 90 % of the new cases of type 1 diabetes are derived, most of whom have the increased risk MHC genotype, selected in this proposal.

In the following years several investigators from other countries, especially Canada and Sweden, joined the Finnish group in the planning of a pilot study. Professor Hans-Michael Dosch of Toronto contributed with immunological expertise and established the contact with the Mead Johnson Nutritionals company, the provider of the study formulas for the first and second pilot studies. Epidemiological, nutritional and clinical input was given in the planning by the Hamilton,Ont., Canada group (Dr. John Vandermeulen, Associate Professors Hertzel Gerstein and Stephanie Atkinson), and also by Professors Gisela Dahlquist of Umeå and Johnny Ludvigsson of Linköping, Sweden. The Finnish group consisted of experts in pediatrics (Professors Hans K.Åkerblom and Mikael Knip, 15 local investigators), including e.g. infant nutrition and pediatric gastroenterology (Dr. Erkki Savilahti ), neonatology (Drs. Anna-Liisa Järvenpää and Martin Renlund ), and in obstetrics ( Professor Kari Teramo ), nutrition science (Dr. Suvi Virtanen ), genetics (Dr. Jorma Ilonen), immunology (Professor Mikael Knip, Dr. Outi Vaarala ), epidemiology (Dr. Antti Reunanen ), and virology (Dr. Heikki Hyöty).

In addition to providing practical information regarding study implementation, valuable data on immunological responses to dietary CM antigens in early infancy and the development of islet-cell specific autoantibodies were obtained. We observed that oral exposure to foreign proteins, i.e. CMPs in infancy resulted in both cellular and humoral immune responses. No responses to ß-lactoglobulin or BSA proteins were seen in infants given a hydrolysed formula. In both groups the immune responses to CMPs declined during a 3-year follow-up suggesting the development of oral tolerance (27, unpublished observations). One infant became positive for IAA before the age of 6 months, with increasing levels later, seroconverted to positivity for ICA and GAD65A between 6 and 9 months and presented with clinical type 1 diabetes at the age of 14 months. He carried the HLA DQB1*0302/x genotype which predisposes to type 1 diabetes and had been given the casein hydrolysate formula as supplementary milk. The appearance of IAA before the age of 6 months and of ICA and GAD65A before 9 months shows that signs of autoimmune â-cell damage may emerge at a very young age. He was the only subject in the total series with signs of a neonatal enterovirus infection, and suffered another enterovirus infection at the end of the first year (28,29). Evidently the dietary manipulation did not prevent this child from contracting type 1 diabetes at an early age, the etiological factor very likely being the neonatal and subsequent enterovirus infection.

These observations lead us to devise a larger, second, multicenter pilot study, aimed besides at testing further the practical implementation of the project and the feasibility of genetic screening, particularly at studying the development of immunological variables during the first 2 years of life.

c.a.b. Second pilot study

The second pilot study was carried out in 15 centres in Finland, the recruitment being initiated in April 1995 and finished in November 1997, the last child reaching thus the age of 2 years in November 1999. The experience and results will be presented briefly below. Smaller series were started later on with EC-support in one center in Sweden, two in Estonia and one in Hungary, and in addition genetic studies and network building in Sardinia, Italy. The results on the type 1 diabetes associated autoantibodies from the other countries are not yet available, as the follow-up is still continuing.

Newborns who had FDRs with type 1 diabetes (i.e. a mother, father or sibling), who met the inclusion and who did not meet the excluson criteria were recruited. The inclusion and exclusion criteria were the same as used in the study proper, presented under d.a., p. 14. To facilitate recruitment and to minimize any possibility of inadvertent exposure to CMP, every attempt was made to identify eligible families before the child was born. Written consent was obtained at this time; the child participated after birth if he/she met the inclusion but did not meet the exclusion criteria. Families not identified until just prior to the onset of maternal labor were approached at that time. Affected mothers were identified during pregnancy via hospitals monitoring preg­nant women with type 1 diabetes. Fathers with type 1 diabetes were identified by a) available history or data already in the medical record of pregnant women, and b) interviewing women at prenatal maternity clinic visits.Pregnant mothers already having one or more children affec­ted by diabetes were identified through various diabetes clinics. Children with type 1 diabetes are usually followed by pediatricians or pediatric endocrinolo­gists, who will be aware of a pregnant mother. These mothers were identified through these physicians.

During the recruitment period of 2 years and 8 months, 521 mothers agreed to join the study. Forty-five newborn infants were excluded, and 476 were subjected to genetic screening and they got a study code at birth. After genetic screening, 234 continued in the intervention study as they had the HLA genotypes conferring increased genetic risk. The drop-out rate over the 2-year follow-up period was 21%, the majority of them occurring before the age of 3 months, i.e. before the first follow-up blood sampling. Therefore we have autoantibody data on 207 children, to be reported below. The reason for dropping out was in about half of the cases some family problem, and the next most common was related to the study formula ( refusal by parents to give or by the child to take it ).

Our co-ordinating staff, the co-ordinating nurse and co-ordinating nutrition advisor have put in a lot of effort to improve the recruitment rate at various hospitals in Finland. It has turned out, that in addition to the initial information to the various parties (nurses, physicians, parents etc.) also repeated newsletters with information and reports on the ongoing study and also site visits are necessary to maintain the recruitment rate. After the 2-year follow-up we have sent out to the participating mothers, a questionnaire asking for their opinions of participating in the study. Only about 54 % have so far responded, and the results are presently being analysed.

The subjects were randomized immediately after birth to define the formula allocation. The randomization code was kept at the data base at the Hospital for Children and Adolescents, University of Helsinki, Helsinki. The recruited subjects were randomi­zed to receive either the test formula ( NutramigenTM, Mead Johnson Nutritionals, USA, not containing intact CMP), or a CMP containing control formula which had an addition of Nutramigen to eliminate taste and smell difference between the two study formulas, whene­ver breast milk was not available. Any subject requiring supple­mental feeding prior to randomi­za­tion (e.g. subjects born at night or on weekends) was given banked breast milk or Nutra­migenTM.

All recruited mothers were encouraged to breast feed; newborn infants were, however, randomi­zed as soon as possible after birth so that any elective formula supplementation or weaning by the mother were done with the appropriate formula. The duration of the intervention was until at least 6 months of age. If the mother chose to exclusive­ly breastfeed up to the age of 6 months (official recommenda­tion of both the Finnish and Swedish Pediatric Societies) she was advised, thereafter, to give the study formula for at least 2 months, i.e. until the age of 8 months. Similarly, if exclusive BF lasted for 5 months, the infant received the study formula until the age of 7 months. Infant feeding practices were altered as little as possible by the trial. In particular, BF practice(s) was entirely at the discretion of participating mothers. However, all relevant data were recorded. Compliance with the denial of CM was monitored by assessing formula usage, regular interviews with participating families, and by appropriate serology.

The overall experience by our staff and participating centers has been positive in relation to the parents' and infants' participation in our study. The various study formulas have been accepted well in general except for a few instances. We have stressed the importance of giving enough time to the mothers at the follow-up visits to discuss the participa­tion, and particularly to relieve possible anxiety triggered by the knowledge of an increased genetic risk in the study subject in families with at least one affected number.

c.a.b.a. Genetic screening

Cord blood was collected at birth in an EDTA-tube, and the sample was forwarded to the genotyping centre in Turku, Finland (Dr. Jorma Ilonen) and screening was performed for the presence of HLA DQB1*0302, DQB1*02, DQB1*0602-03, and DQB1*0301. Results were sent to the data centre in Helsinki, Finland within 5-7 days.Only subjects who were HLA DQB1*0302 and/or DQB1*02-positive and HLA DQB1*0602, *0603 and *0301-negative were invited to continue in the study; all other subjects (approxi­mately 50% of randomized subjects) were withdrawn from the study at that time; their parents were told that genetic screening suggests that their child is at low risk for type 1 diabetes. The distribution of HLA -genotypes in the two groups was as follows: DQB1* 02/*0302 22%, *0302/x 39%, and *02/y in 39% in the CM-based formula group, and 21%, 41%, and 38% in the casein hydrolysate group, respectively.

The distribution ofDQB1 genotypesin Finnish familial cases is shown in Table 1. This suggests that the sensitivity of the used selection criteria is 84.6%,when 50.7% of the studied newborns can be excluded from the study. In terms of odds ratios the subjects with selected risk genotypes had an OR value of 5.7 compared to 0.2 of nonselected. When the general familial risk of 3% by the age of 10 years is used as the basis of the calculation, the predictive positive value shows that 5.4 % of selected children could be expected to develop diabetes compared to only 0.8 % within the nonselected group.

Table 1. HLA-DQB1 genotypes in Finnish newborn children recruited to the TRIGR study compared to familial type 1 diabetes cases.

*Data from reference 30

To ensure the usefulness of genetic screening also in the other participating populations type 1 diabetic children andcontrol subjects from Estonia and Sardinia were analysed with the same screening method (Table 2). Information on the genotypes in Hungary was obtained from the Medical University of Pecs (Dr Robert Hermann).

The sensitivity was highest in Sardinia but the number of DQB1*02 positive subjects in the control population is very high there making the screening system relatively unspecific. The peculiar characteristic of the Sardinian population was also very clear when the effect of adding a further step to the screening procedure was tested in samples from different populations. This was made by typing series of samples with DQB1*02/x (x#*0301,*0302,*0602,*0603) genotype for the presence of DQA1*05 (DR3 haplotype) or DQB1*0201 (DR7 haplotype) with an additional PCR and hybridisation reaction. Also in the control population the frequency of DR3 haplotype was very high in Sardinia and and DR7 haplotype was very rare making the value of DQA1 typing useless (Table 2). The additional value obtained was also limited in the Finnish population, because the prevalence ofthe DR7 haplotype was rather low and its effect neutral. Instead, in the Estonian and Hungarian populations this testing step seems to be very useful because of the higher RR value associated with the DR3 haplotype and especially also because the DR7 haplotype in these populations seems to be protective.

Table 2. The risk carried by the by HLA-DQB1*02/x genotype when divided according to the presence of DQA1*05 and/or DQA1*0201 alleles in the haplotype

*Hermann et al. Unpublished data

The additional value of DQA typing in selected cases was also demonstrated in Russian and Latvian populations (31).

Based on these studies we have decided that the study proper will be conducted using a two phase screening where HLA-DQB1 genotyping is the first phase. Those with DQB1*02/*0302 and DQB1*0302/x (x¹*02,*0301 or *0602) genotype are directly selectedfor the study and those with DQB1*02/y (y¹*0301,*0302, 0602 or 603) genotype are analysed for the presence of DQBA1* alleles DQA1*05 and DQA1*0201. Those who are positive for DQA1*05 but not for DQA1*0201 are also selected to continue in the study group.

During the second pilot study we established logistics for sample distribution and swift information transfer between participating hospitals and the laboratory. The DNA hybridisation based method does not have any strict requirements for howthe samples should be sent but the main consideration is the need for rapid genotyping. The use of courier delivery of samples and e-mail or fax in transferring the genotyping result was observed to ensure smooth data processing also in Estonian, Hungarian and Swedish centers participating in the project.

Islet-cell related autoimmune markers were analysed in the Research Laboratory, Department of Pediatrics, University of Oulu under the supervision of Professor Mikael Knip. The methodology for the assays of islet cell antibodies (ICA), insulin antibodies (IAA), antibodies to the 65kD isoform of glutamic acid decarboxylase (GADA) and antibodies to the protein tyrosine phosphatase related IA-2 molecule (IA-2A) was the same as described in the present application under d. RESEARCH DESIGN AND METHODS, d.i.b. Methodology for Humoral Markers of Beta-Cell Autoimmunity.

 The results from the 207 children in Finland are summarized in Table 3.

Table 3. Emergence of ICA, IAA, IA-2A and GADA by the age of 2 years (n=207)

A decrease in the frequency of autoantibodies in the casein hydrolysate vs. control formula group occurredfor all variables except for GADA, the relative decreases being: ICA 63%, IAA 51%, IA-2A 64%, at least one autoantibody 60% and at least two autoantibodies 44%.

Theeffect of the intervention on the risk of seroconversion of any of the four autoantibodies was as follows: casein hydrolysate vs. CM-based formula, risk ratio (RR) 0.40 (95% CI 0.13-1.03), p=0.059. As there were some differences between the two groups in the duration of BF and formula feeding, the analysis was done by adjusting for the duration of study formula feeding, the RR being 0.34 (0.11- 0.89), p=0.027.
(Here a summary of the CM antibody findings from Dr.Erkki Savilahti, when the results are available in March 2000).

c.a.b.d. Child health

The children and their mothers visited the outpatient clinics when the child was 3, 6, 9, 12, 18 and 24 months old, being seen by the study nurse or nutrition advisor, and at certain intervals by the pediatrician. A physical examination was performed, and a venous blood specimen was obtained after using a local analgesic ointment for venipuncture. The children's health was in general good. The height and weight development in the two groups was similar.

By now, after analysing the type 1 diabetes associated autoantibody data, we can rule out option 3, i.e. we have not done harm to the investigated children. We feel strongly that we have the justification and obligation to continue into the study proper phase with the purpose to get a final answer to the study question mentioned under a. SPECIFIC AIMS , p.1.

c.b. Other studies related to the CM and type 1 diabetes hypothesis, and to the prediction of type 1 diabetes

The undersigned P.I. and his associates have over the last 15 years conducted several studies with relevance to the CM and type 1 diabetes hypothesis. Among them several came from the nationwide "Childhood Diabetes in Finland" (DiMe) Study, aimed at elucidating the genetic, immunological and environmental determinants of type 1 diabetes in children in Finland, the undersigned P.I. being the Project Director and one of the two P.I.'s of the DiMe-project. The population-based DiMe-study comprised 750 consecutively diagnosed 0-14-year-old children with newly diagnosed type 1 diabetes, 819 3-19-year-old non-diabetic siblings, and control subjects (34).

c.b.a.Infant feeding studies

In the DiMe-study Virtanen et al. showed an inverse association between the duration of BF and risk of type 1 diabetes in 0-6-year-old (35) and 7-14-year-old (36) subjects. We were the first to show that after adjustment for duration of BF, an increased risk of type 1 diabetes in conjuction with early introduction of dairy products was still present (5).

As others have proposed that an increased weight gain in infancy may act as a risk factor for type 1 diabetes in children, Hyppönen et al. (37) analysed weight measurements from the 1st year of life of 435 full-term subjects with type 1 diabetes and 386 control subjects. The results indicated that an early exposure to CM formula feeding and rapid growth in infancy are independent risk factors of childhood type 1 diabetes.

c.b.b. Studies on the role of CMPs in the pathogenesis of type 1 diabetes

Savilahti et al. of our group in Helsinki were the first to report in man on elevated levels of CM and b-lactoglobulin antibodies at the diagnosis of type 1 diabetes (38). This finding was later confirmed in the DiMe-study, comprising 706 children with newly diagnosed type 1 diabetes, 456 non-diabetic siblings, and 105 unrelated age-matched controls below 7 years of age (39). Multivariate analyses including infant feeding and milk consumption variables, current age and ICA status indicated that young age at the introduction of dairy products and high CM consumption during childhood inrease the levels of CM antibodies and that high IgA CM antibodies are independently associated with an increased risk of type 1 diabetes (40). Features of our data suggest that antibodies to CM and b-lactoglobulin are not derived from the autoimmune process probably responsible for destroying ß-cells and causing type 1 diabetes. Instead, we inferred that a high titre of CM antibodies is an epiphenomenon of an insult and immunization caused by CMPs, which in some genetically susceptible individuals may lead to type 1 diabetes after a long but variable time interval (39).

We have also tested the cellular immunity to several CMPs (b-lactoglobulin, BSA, a-casein, ovalbumin) in patients with newly diagnosed type 1 diabetes, and only T-cell reactivity to b-lactoglobulin was enhanced in affected patients when compared to the non-diabetic children (20).

We have studied the role of the gut immune system in type 1 diabetes by analysing the expression of a gut-specific adhesion molecule (a4b7-integrin) on the islet cell antigen (GAD)-reactive lymphocytes from patients with type 1 diabetes (21).We found a marked decrease in proliferation of PBMC to GAD in four of seven patients with type 1 diabetes, whereas the proliferation response to a parenteral antigen, tetanus toxoid increased, when a4b7-integrin-positive cells were depleted. These findings demonstrate that islet cell-reactive lymphocytes express a gut-specific homing receptor,which emphasizes the role of gut immunity in type 1 diabetes. This regulatory defect in the gut lymphocyte population may be responsible not only for b-cell reactivity but also for enhanced reactivity against oral antigens, e.g. CMPs (21). We also observed that orally fed CMPs induce an elevation in soluble intercellular adhesion molecule 1 (ICAM-1) in infants, possibly reflecting the generation of an immune response against these proteins, because ICAM-1 has an important role in lymphocyte activation (41).These observations, and the findings on humoral and cellular immunity to dietary CMPs can be interpreted as indicative of dysregulated oral tolerance. This regulatory defect of the gut immune system may actually play a fundamental role in the pathogenetic process leading to type 1 diabetes, as proposed earlier by Vaarala et al. in our group (20,21).

Vaarala et al. have recently generated evidence for a new possibility to explain the epidemiological link between the risk of type 1 diabetes and early exposure to CM containing formulas, i.e. by immunization to bovine insulin (42,43). It is known that CM products contain bovine insulin. Initially Vaarala et al. observed that CM-based formula could induce insulin-binding antibodies in children (42). Thereupon, in a larger series it was found that the amount of IgG-antibodies binding to bovine insulin was higher at 3 months of age in infants who were exposed to CM-based formula thanin infants who were exclusively breast-fed at that age ( p< 0.0001) (43). In that series there were 11 subjects who developed signs of b-cell autoimmunity before the age of 2 years. Those children had increased levels ofbovine insulin antibodies over time in contrast to the other children whose antibody levels to bovine insulin decreased after the age of 12 months. These observations imply that the immune response initially induced by bovine insulin may later be diverted into autoaggressive immunity against the b-cells in a few unfortunate individuals, i.e. those who subsequently experience b-cell damage and progress to clinical type 1 diabetes.

c.b.c. Studies on serological risk markers and prediction of type 1 diabetes

The results of the present project are depending not only on the amount of manifest type 1 diabetes cases in the intervention and control groups, but also on the appearance of "surrogate" markers of the disease, i.e. type 1 diabetes associated autoantibodies. Therefore it is relevant to describe below briefly studies of our group on these markers, important in the prediction of the disease.

Knip et al. described the natural history of preclinical type 1 diabetes in a group of 57 siblings positive for ICA and/or IAA when first screened within 6 months after the diagnosis of the proband (44). Those who progressed to clinical diabetes were characterized by young age, strong and increasing signs of islet-cell specific autoimmunity, reduced insulin secretory capacity and emerging glucose intolerance. Kulmala et al. evaluated the predictive characteristics of ICA, IAA, GADA and antibodies to IA-2A in an unselected population of 755 siblings of children with type 1 diabetes, 32 of whom progressed to clinical disease within 7.7 years of the initial sample taken (45). The risk for type 1 diabetes in siblings with four, three, two, one, or no antibodies was 40, 70, 25, 2 and 0,8 %, respectively. The results indicated that the combined screening for IA-2A and GADA may replace the ICA assay, giving comparable sensitivity, specificity, and positive predictive value. In another study on the same series, Mrena et al. evaluated the efficacy of grading the siblings into different risk categories based on the number of positive antibodies alone, or in combination with the first-phase insulin response FPIR (46). The results indicated that it is possible to grade siblings of children with newly diagnosed type 1 diabetes into categories with significant differences in the subsequent risk of clinical disease and time to diagnosis.

Kulmala et al. studied the relationships between genetic markers and disease-associated autoantibodies in an unselected population of 701 siblings in the DiMe-study over a period of 9 years (47). The combination of the genetic and immunological markers increased the positive predictivevalues of all autoantibodies substantially. Because the combination also resulted in reduced sensitivity, autoantibodies alone were recommended as the first-line screening in siblings. Finally, Kimpimäki et al. evaluated the emergence of diabetes-associated autoantibodies in 180 initially unaffected siblings, younger than 6 years at the initial sampling. Fifteen siblings (18%) manifested type 1 diabetes before the age of 10 years. All progressors had increased HLA-defined genetic susceptibility . Thirteen of the 17 siblings testing positive fortwo or more autoantibodies before the age of 6 years became diabetic before the age of 10 years (48). These findings have been utilized in the power calculations of the study proper phase in the present application.

c.c. Rewiew articles on environmental factors in type 1 diabetes and prevention trials

The undersigned P.I. has with his associates in recent years reviewed the putative role of various environmental factors, and particularly the role of CMPs in the etiology of type 1 diabetes (49,7,50-52), the interaction of genetic and environmental factors in the pathogenesis of the disease (11), participated in the discussion on the role of CMPs in the pathogenesis of type 1 diabetes (53,54), and reviewed critically the type 1 diabetes prevention trials in progress (55). Some of these articles are enclosed as Appendix:

d.RESEARCH DESIGN AND METHODS

d.a.Sample Specification

Newborn infants who have FDRs with type 1 diabetes (i.e. a mother, father or sibling), and who meet the inclusion but not the exclusion criteria will be recruited: