Final Report Summary - DIABIMMUNE (Pathogenesis of Type 1 Diabetes - Testing the Hygiene Hypothesis)
Executive Summary:
Preliminary data indicate that there is a strong association between the incidence of immune-mediated diseases and improving standard of living and hygiene. One of the steepest gradients in standard of living worldwide is present at the border between Russian Karelia and Finland with a sevenfold difference in the gross national product, while Estonia represents a country in rapid transition. These three populations comprise a “living laboratory” providing a unique possibility to test the hygiene hypothesis and gene-environmental interactions in the development of immune-mediated diseases. According to the hygiene hypothesis there is an association between the increased incidence and prevalence of immune-mediated diseases and decreased exposure to pathogens. The incidence of T1D is six times lower in Russian Karelia than in Finland, whereas there are very limited differences in the frequency of predisposing and protective HLA (human leukocyte antigen) genotypes in the background population. The aim of the DIABIMMUNE project is to assess the role of the hygiene hypothesis in the development of immune-mediated diseases, T1D in particular, and to define the mechanisms behind the potential protective effect conferred by microbial agents. The objective was to study newborn infants with increased genetic risk to type 1 diabetes and to follow children at the age of 3 and 5 years in Finland, Russian Karelia and Estonia. The children are tested for organ-specific autoantibodies, allergies, infections, gut microflora, and for nutritional factors.
Extensive biobanks comprising genetic samples, serum samples, viral and allercological samples as well as stool samples from study subjects have been established in the DIABIMMUNE project. This material has been used to study the disease process resulting in overt type 1 diabetes or any allergic disease. A strongly protected real-time database including all information collected and all research data generated has been established. Altogether 8863 families gave informed consent to genetic screening of their newborn infant. By the end of February, 2014 altogether 3740 families have attended the first study centre visit with their 3-year old child in Finland, Estonia and Russian Karelia and 7250 follow-up visits for the young children cohort have been performed in the DIABIMMUNE study centres.
Altogether 14.689 HLA samples have been genotypes in the laboratory of University of Turku. Analyses of diabetes-associated autoantibodies [against insulin (IAA), glutamic acid decarboxylase (GADA), insulinoma-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A)] have been performed in 13,335 samples in the autoantibody laboratory, University of Helsinki. The coeliac disease marker antibodies (IgA-TTGA with EliA or/and ELISA, IgG-TTGA, IgG-DGPA, EMA) have been tested in 16,403 blood samples. National Institute for Welfare and Health has received altogether 581 peripheral blood samples from children participating in the birth cohort arm and 341 samples from the cohort of young children. Flow cytometry studies, T-cell suppression tests or phenotyping of regulatory T-cells have been performed from the samples. University of Turku has received 1628 Tenmpus RNA samples from the participants in the birth cohort and 1632 from participants in the young children cohort for RNA isolation and quality control. Four hundred twenty-three samples have been processed for RNA isolation and genomic DNA. The work package of natural history of allergy has produced the knowledge of frequencies of allergic diseases and atopic sensitisation in cohorts of Finnish, Estonian, and Russian Karelian children. These studies have shown that atopic sensitisation and allergic diseases are definitely more common in Finnish children followed by Estonian children, while Russian Karelian children have less sensitisation and allergy. University of Tampere has analysed the frequency of virus infections in the three populations by detecting selected viruses and parasites in stool samples. In addition, selected respiratory viruses were analysed from nasal swab samples. The data in the gut microflora studies analysed by the Academic Hospital Groningen shows that gut colonization of newborn infants is related to the duration and exclusivity of breastfeeding. Especially the colonization with specific bifidobacterial species showed a relation with autoantibody positivity and type 1 diabetes. University of Tampere is responsible for the dietary studies. Altogether 1829 3-day food records have been collected in the birth cohort and in the young children cohort for further analysis.
The DIABIMMUNE project represents translational research with a strong clinical component. The study cohorts comprise children and addresses common immune-mediated diseases, presenting in a majority of the affected cases in childhood, but with a life-long duration as chronic disorders. The study focuses on early events in the development of type 1 diabetes. The DIABIMMUNE project has important socio-economic aspects. The incidence of type 1 diabetes has increased several fold in most industrialised countries over the last 50 years. Accordingly there is a definite need of effective strategies to stop the increase in disease incidence and the ultimate aim would be to turn the trend around, i.e. to effectively decrease the incidence of type 1 diabetes in the developed countries. The data generated so far supports the hygiene hypothesis but it is too early to conclude whether the total microbial load or specific microbes represent the critical culprit.
Project Context and Objectives:
The hygiene hypothesis postulates a relationship between the increasing incidence and prevalence of immune-mediated diseases in modern society and a childhood environment resulting in a decreased pathogen exposure. The hypothesis was initially introduced to explain the increasing frequency of allergy and asthma in developed countries. Recently the hygiene hypothesis has been implicated to contribute to the increasing incidence of autoimmune diseases such as type 1 diabetes (T1D).
The overall objective of the DIABIMMUNE Project has been to test the hygiene hypothesis in T1D and delineate the mechanisms mediating the influence of the standard of hygiene and living on the immune system and leading to a dysregulated immune system predisposing to T1D and allergy.
The specific scientific objectives are
a) to characterise the natural history of beta-cell autoimmunity in three cohorts of young children(Estonia, Finland and Russian Karelia) exposed to contrasting standards of hygiene and living;
b) to identify environmental triggers and determinants of beta-cell autoimmunity;
c) to define gene-environmental interactions in the emergence of signs of beta-cell autoimmunity;
d) to delineate the ontogeny of the immune system in infants and young children living in three contrasting environments by using modern tools of functional genomics;
e) to define the maturation and function of regulatory T cells in infants exposed to contrasting standard of hygiene and living;
f) to analyse the impact of gut microbial colonisation on the ontogeny of the immune system in young infants;
g) to assess the effect of various dietary factors on gut microbial flora and on immune responses induced by acute microbial infections;
h) to define the role of gut microbial flora, acute microbial infections and consumption of foods affecting these microbes in the development of T1D; and
i) to compare the above mentioned variables between children who develop beta-cell autoimmunity and those who present with allergy to identify possible common ethiopathogenetic factors and immunopathogenetic pathways.
The DIABIMMUNE Project has had the following technological objectives:
(i) to establish a biobank comprising genetic samples (DNA and dry blood spots) from newborn infants in Finland, Estonia and Russian Karelia and at least 3500 3-year-old children and genetic samples from 3500 nuclear families (as a minimum a trio including the father, mother and the index cases) from Finland, Estonia and Russian Karelia;
(ii)to screen 6660 newborn infants and 3500 3-year-old children for HLA-conferred susceptibility to T1D and autoimmunity;
(iii)to establish a biobank comprising at least 960 individuals with sequential biological samples taken at intervals of 1-6 months starting from birth. These samples include serum, peripheral blood mononuclear cells, rectal swabs and stools. In addition RNA samples from peripheral blood are collected and stored from a smaller cohort, and breast milk samples are collected from lactating mothers when the index case is 3-month-old;
Project Results:
During the 5 years of the study a lot of data has been collected and the novel knowledge has been generated and continues to be genrated while the data collection is still continuing in Russian Karelia due to the delayed recruitment of the 3-year-old children in the young children cohort. Most analyses are also still continuing.
Extensive biobanks comprising genetic samples from subjects living in three different countries, Finland, Estonia and Russian Karelia, has been established in DIABIMMUNE project. These biobanks can be used to assess the role of genetic elements involved in the development of organ-specific autoimmune and allergic diseases. The project has resulted in a series of other biobanks comprising different biological materials from the subjects taking part in the surveillance part of the project. Such material can be used to study the disease process resulting in overt type 1 diabetes (T1d) or any allergic disease.
A strongly protected real-time database including all information collected and all research data generated has been established starting from the inclusion of the first index case into the study until the last follow-up visit of the study. This database has been utilised for monitoring the progress of the study and for data analysis related to the objectives of the study. The identification of the study participants is based on individual codes in the central database without using personal identifiers. In addition there is in each recruiting study centre a free-standing database combining the individual study code with data identifying the person behind the code.
The recruitment for the birth cohort started in September 2008 and was completed in Finland and Estonia in May 2010 and in Russian Karelia in March 2011. There was a need to extend the recruitment in Russian Karelia due to the lower than expected consent rate to the follow-up among families with an HLA-eligible baby. By the end of February, 2014 altogether 8863 families gave informed consent to genetic screening of their newborn infant. Almost 19% (1653; 18.6 %) of the infants screened were observed to carry HLA-conferred susceptibility to autoimmunity, which is very close to the expected proportion of 18%. Eight hundred twenty-nine families have decided to join the follow-up study of HLA-eligible subjects in the birth cohort. Altogether 4003 follow-up visits have been performed for the birth cohort children in the DIABIMMUNE study centres. The recruitment for the young children cohort started in the fall 2009 in Espoo, Finland and in Tartu, Estonia. Petrozavodsk, Russian Karelia started in October 2011. By the end of February, 2014 3740 families have attended the first study centre visit with their 3-year-old child. The recruitment was closed in June 2011 in Tartu with 1681 families participating and in July 2011 in Espoo with 1575 families. In Petrozavodsk, Russian Karelia the recruitment for the young children cohort was finalized in March 2014 with 484 families. Altogether 7250 follow-up visits have been performed for the young children cohort in the DIABIMMUNE study centres. The last follow-up visits were conducted in Espoo, Finland in June 2013 and in Tartu, Estonia in September 2013. The follow-up in Russian Karelia is still going on.
The HLA laboratory in Turku, Finland, has worked effectively from September, 2008. Altogether 14,689 HLA samples have been analysed by the end of February 2014.
The analyses of diabetes-associated autoantibodies [against insulin (IAA), glutamic acid decarboxylase (GADA), insulinoma-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A)] have been performed in the autoantibody laboratory, University of Helsinki. In the young children cohort, six Finnish and four Estonian children have so far presented with clinical T1D. All affected children have tested positive for multiple autoantibodies during the follow-up. Progression to T1D was twice as common in Finnish children as in Estonian children, even though, due to the low numbers, the difference was not statistically significant. The fact that no case of T1D hase been identified in Russian children thus far is within the expected incidence range characteristic of the paediatric population of Russian Karelia.
The coeliac disease marker antibodies (IgA-TTGA with EliA or/and ELISA, IgG-TTGA, IgG-DGPA, EMA) have been tested in 16,403 blood samples. The IgA-TTGA levels ranged from 0 to 4262 U/ml, IgG-TTGA levels between 0 and 113 U/ml, and IgG-DGPA levels between 0 and 245 U/ml. There was a suspicion of IgA deficiency in 19 cases, but all serum samples were IgG-TTGA and IgG-DGPA negative. The serum samples from the birth cohort children comprised a umbilical cord blood sample sand serum samples taken at 3, 6, 12, 18, 24 and 36 months of age. Altogether nine children had developed IgA-TTGA during the 3-year follow up. In all nine children coeliac disease was confirmed by histological examination of small bowel biopsies. Seven (1.8 %) children out of 387 children from Espoo and two (0.61 %) out of 330 children from Tartu enrolled in follow-up tested positive for IgG-TTGA in cord blood.
The coeliac disease marker antibodies in the serum samples from the children aged 3, 4 and 5 years (n=6868) in the young children cohort and from their siblings (n=755) and parents (n=143) were also analysed. Combining the results in the index childrem we discovered that 21 (1.3 %) children in the original Espoo (n=1575) and 14 (0.8 %) children in the original Tartu cohort (n=1681) have tested positive for IgA-TTGA once or several times by 5 years of age. In the Petrozavodsk cohort (n=310) two children (0.65%) turned out to have coeliac disease associated antibodies. Based on these results the children were invited to gastroduodenoscopy and small bowel biopsy. Altogether, 22 children were biopsied and coeliac disease was histologically confirmed in 19 (13 from Espoo and six from Tartu). In three children coeliac disease was not detected in spite of the presence of IgA-TTGA. Among these children one had shown repeatedly high IgA-TTGA values, one had moderately elevated antibody levels and one tested only once weakly positive.
The National Institute for Welfare and Health has received altogether 581 peripheral blood mononuclear cell samples from children participating in the birth cohort arm and 341 samples from the cohort of young children by the end of February 2014. Flow cytometry studies, T-cell suppression tests or phenotyping of regulatory T-cells have been performed from the samples. In the T-cell studies has been compared The number and function of regulatory T cells have been compared between Estonian and Finnish children in the T-cell studies. No fresh blood samples have been received from Russian Karelia due to problems with the approval of the transportation of biological samples from Russian Federation and the impossibility to arrange the sample delivery to Helsinki, Finland within 24 hours after the obtaining fresh T-cell samples in Petrozavodsk.
The observation of increased numbers of highly activated Treg cells with high expression of FOXP3 at 3 months of age in Estonian children suggest that regulatory mechanisms are more active after birth in Estonian than Finnish children. This could be due to differences in the microbial load between the two countries and the analyses of the association of gut microbiota and Treg cells, which are on-going, may reveal bacterial strains or species with potential therapeutic possibilities for the activation of T-regs. The findings of regulatory T-cell studies show that the emergence of a subpopulation of highly activated Tregs is seen when the child’s immune system matures during the first 3 years of life
University of Turku has received 1628 follow-up Tempus RNA samples from the birth cohort children and 1632 samples from the young children cohort by the end of February 2014 for RNA isolation and quality control. Four hundred twenty-three samples have been processed for RNA isolation and genomic DNA. Data generated for functional genomics has produced novel information on the effect on the standard of hygiene on the expression of critical molecules affecting the immune system. The transcriptomics measurements of the cord blood samples collected at the time of birth clearly indicated, that the children born in Petrozavodsk, Russian Karelia, deviate from children born in Espoo, Finland or Tarto, Estonia. The results suggest that newborn infants in Russian Karelia have been exposed to TLR ligands in utero, and that the subsequent immune response is developmentally more mature. Therefore, lack of in utero immunomodulation may influence the predisposition towards immune mediated diseases in the Westernized world. These data will lead to further studies focusing on the in utero period in the regulation of maturation and activation of the immune system. The results have been communicated to the scientific community through poster presentations in two international scientific meetings, and a manuscript describing the results has been submitted for publication in an international peer-reviewed scientific journal. In addition, further investigations and analyses ongoing in the Work package 6 will focus on investigating the transcriptomics changes occuring A) during immune system maturation (3 months -3 years), and B) during appearance of T1D-associated autoantibodies (Birth cohort and Young Children Cohort). The results will be published as separate articles im scientific journals.
The work package of natural history of allergy has produced the knowledge of frequencies of allergic diseases and sensitisation in cohorts of Finnish, Estonian, and Russian Karelian children born between 2005 and 2011. The allergy studies have shown clear differences between the Estonian, Finnish and Russian Karelian children. The allergic sensitisation is less prevalent in Estonian and Russian Karelian children in the birth cohort than in the Finnish peers. Similar results were observed seen among 3-5-year-old children in Finland and in Estonia.
University of Tampere has continued to develop new technologies for screening of microbes from stool, serum and nasal swab samples and carried out analyses of samples collected. The frequency of virus infections was analysed in the three populations by detecting selected viruses and parasites in stool samples. In addition, selected respiratory viruses were analysed from nasal swab samples. Enteroviruses were found to be most frequent in young children in Russian Karelia, followed by Estonian children, while they were detected more rarely in Finnish children. However, norovirus and parechovirus infections did not differ between the countries and rhinovirus infections were most frequent in Finnish and Estonian children and less frequent in Russian Karelian children. The data generated in the work package of microbial infections indicates that virus infections have an effect of immune regulation and development of IgE-mediated sensitization. This offers possibilites to use these viruses or their immunologically acitive structural components in the development of new treatments for allergic diseases.
The goal of Academic Hospital Groningen has been to delineate the gut microbial colonization in infants exposed to contrasting standards of hygiene and to investigate which specific intestinal microbes are involved in the development of T1D and other immune-mediated diseases in 3 and 5-year old children. The data generated by the gut microflora studies show that the intestinal colonization of newborn infants is related to the duration and exclusivity of breastfeeding. Especially the colonization with specific bifidobacterial species showed a relation with autoantibody positivity and T1D. The colonization after weaning showed that higher numbers of the Bacteroides genus, a lower abundance of species from Clostridium cluster IV, a lower abundance of particular butyrate producers from Clostridium cluster XIVa and fewer Prevotella were present in Finland. Finnish children had a different microbiota than the Estonian children who are characterised by a flora that is now generally assumed to be more protective.
The work plan included more detailed HLA genotyping and genotyping for major T1D associated non-HLA genes including INS, PTPN22 and CTLA4 in the subgroups of children developing beta-cell specific autoimmunity. This has not been yet performed as the final autoantibody results have just become available and the control group for children with autoimmunity has not yet been selected.
University of Tampere is also responsible for the dietary studies. Altogether 1829 3-day food records have been collected in the birth cohort and in the young children cohort for further analysis. Among children 0-36 months of age, 3-day food records have been collected at the 6 and 12-month visits and among children 3-5 years of age at the 4 and 5-year visits. The food database is completed for all the foods used in the DIABIMMUNE study and the food data entry of the food records is also completed in Finland and in Estonia. In Russia, the collection of food records has been challenging, and the collection continues. The analyses of the dietary data are continuing.
DIABIMMUNE Project has not received all its milestones due to the difficulties and the challenges with the recruitment and the follow-up in Petrozavodsk, Russian Karelia. The follow-up visits of the young children cohort are continuing to be performed in Russian Karelia until the end of the year 2015. The data analyses are also continuing due to the delay and the challenges in the recruitment and the follow-up of Russian Karelian children. The continuation of the study has been made possible by the separate funding received from Academy of Finland, University of Helsinki and Sigrid Juselius Foundation. The DIABIMMUNE study has generated data that can be utilized as background information in the part of the INTERCONNECT project focusing on type 1 diabetes.
Potential Impact:
The study setting has provided a unique opportunity to define the impact of contrasting standards of hygiene and living on the appearance of signs of beta-cell autoimmunity and allergy in young children. The project has generated and is still generating novel knowledge that can be exploited for developing preventive strategies that are able to break the present alarming trend of increasing incidences and prevalences of type 1 diabetes seen in most EU countries. Similar contrasts in standards of hygiene and living can hardly be found anywhere else in Europe or even globally. Finland represents in relative terms a highly developed country, Russian Karelia a so far poorly developed country and Estonia a country in rapid transition from poverty to relative prosperity. The adjacent location of the three countries standardises most geophysical factors, such as temperature, rain, and hours of sunshine and these factors cannot explain the differences observed e.g. in the incidence of type 1 diabetes (T1D) between Finland and Russian Karelia in our previous study. In addition Finns, Karelians and Estonians are genetically related providing an opportunity to assess the direct impact of environmental factors. In Russian Karelia Russians comprise the majority of the population, but the proportion of Karelians is about 12%. In Estonia approximately 26% of the population is of Russian origin making it possible to perform comparative analyses between children of Estonian or Russian origin, both groups born in Estonia.
DIABIMMUNE project represents translational research with a strong clinical component. The study cohorts comprise children and addresses common immune-mediated diseases, presenting in a majority of the affected cases in childhood, but with a life-long duration as chronic disorders. The proposal focuses on early events in the development of T1D by studying children with HLA-conferred disease susceptibility from birth up to the age of 3 years. In addition a genetically unselected, more extensive cohort including 3-year-old children has been recruited and studied. The latter cohort was re-examined 2 years later to generate additional information on the natural history of preclinical T1D and allergy. A multidisciplinary approach was applied as the consortium comprises diabetologists, allergy specialists, virologists, immunologists, experts on infectious diseases, and nutritional scientists. The longer-term goal of the project is to develop intervention strategies mimicking the mechanisms identified behind the protective effect conferred by the Russian-Karelian environment against T1D and other immune-mediated diseases. If the hygiene hypothesis is confirmed, oral administration of safe microbial products (live vaccines and probiotic-type bacterial products), provides a tempting option for a future intervention trial in populations with a high incidence of T1D and other immune-mediated diseases. Microbial products including live vaccines and bacterial components have already been licensed for medical use in Europe.
The DIABIMMUNE project has important socio-economic aspects. The incidence of T1D has increased several fold in most industrialised countries over the last 50 years. Accordingly there is a definite need of effective strategies to stop the increase in disease incidence and the ultimate aim would be to turn the trend around, i.e. to effectively decrease the incidence of T1D in the developed countries. Such strategies require, however, novel knowledge of those factors, which have contributed to the increase in incidence rate of T1D and other autoimmune diseases in Western countries after World War II. The DIABIMMUNE project provides a unique opportunity to generate such new knowledge, since the study has made it possible to compare the maturation of the immune system in young children representing populations with conspicuous contrasts in their standard of hygiene and living. Simultaneously it will be possible to time the autoimmune and allergic process and thereby facilitate the identification of host-related and environmental factors functioning as triggers of the disease process and as determinants of the progression from the initiation of the process to full-blown clinical disease.
T1D is an immune-mediated disease where the destruction of the insulin-producing beta cells is triggered by some so far unidentified exogenous factor(s) in an individual with increased genetic disease susceptibility. The diagnosis of clinical diabetes is preceded by an asymptomatic preclinical period, which may last for months and years. The shortest prediabetic process that we have observed in prospective studies based on the occurrence of diabetes-associated autoantibodies in the peripheral circulation lasted for less than 3 months and the longest for more than 20 years. Currently T1D is an incurable chronic disease. Despite progress in insulin therapy and other aspects of diabetes care over the last 30 years, only a small minority of the affected patients are able to maintain good metabolic control. As a consequence the disease may lead to a series of complications, such as retinopathy, nephropathy and neuropathy resulting in decreased quality of life and reduced life expectancy. From the social point of view T1D is associated with substantial costs. It has been estimated that presentation of T1D in childhood results in Finland in additional lifetime costs amounting to about 1 M€.
Allergic diseases are the most common group of chronic disorders in children and adolescents in the developed countries. The incidence and prevalence of such diseases have increased substantially after World War II in all EU countries. The European Allergy White Paper from 1997 states that allergic diseases have reached epidemic proportions and recognises that these diseases are a public health problem in Europe. The paper also asks for effective preventive measures and emphasises that prevention requires more precise identification of atopic individuals and risk factors in the aetiology of atopic diseases. One aim of the DIABIMMUNE project has been the identification of such risk factors and possible protective factors that can be utilised in the development of effective preventive strategies.
Preventive aspects
The results of this project can potentially be exploited to establish strategies aimed at delaying or preventing progression to clinical T1D and at decreasing the incidence and prevalence of allergy in developed countries, such as the EU countries. By studying the natural history of beta-cell autoimmunity in three countries with a strong gradient in the rate of T1D we have learned more about at which age the first autoantibodies appear and about the rate of progression to clinical disease. Such information can be utilised to assess the window open for primary prevention of T1D. In addition it has provided an opportunity to identify exogenous triggers of beta-cell autoimmunity. The study also facilitates the identification of exogenous factors affecting the progression to clinical disease. The identification of such factors might open up new avenues for preventive measures.
Observation of the natural history of allergy provides information on at which age the immune system is diverted into reactivity favouring allergic responses. This knowledge defines the optimal window for intervention aimed at preventing such diversions. The study setting has also provided opportunities to identify host-related and environmental factors and their mutual interactions triggering allergy and factors potentiating the development of asthma and other allergic diseases. Also factors protecting against allergic diseases might be identified.
Scientific and health-related aspects
It has been shown previously that HLA-conferred susceptibility to T1Dis a strong determinant of the appearance of signs of beta-cell autoimmunity in young children. The DIABIMMUNE project has facilitated the analysis of gene-environment interactions in the emergence of signs of beta-cell autoimmunity and allergy by introducing more heterogeneity both in relation to genetic and environmental factors compared to a birth cohort study implemented in a single country.
By comparing the expression profile of immunologically active molecules with 3-6-month intervals during the first year of life starting from birth between infants living in the three countries taking part in the study it is possible to define the impact of the contrasting standard of hygiene and living on the expression profiles allowing the identification of changes over time. Such expression profiles may generate novel hypotheses that might be further explored based on the various forms of biological material collected from the study subjects.
The study protocol facilitates the assessment of the impact of dietary factors on the maturation of the immune system. This generates novel knowledge that may be used to optimise both early infant nutrition and subsequent diet to support the establishment of normal immune responses. The study design has also allowed a comparison of the colonisation of the gastrointestinal tract in infancy between subjects from the three countries and the analysis of the effect of dietary factors in this context. The interactions between gut microbial flora, acute microbial infections and dietary factors in the development of organ-specific autoimmunity and allergy can be analysed in a versatile way due to the substantial variation in these factors expected to be seen between subjects from the three countries.
We have studied early infant nutrition and the diet in young children within the framework of the current project. This provides novel information on the diet of infants and young children in Estonia and in Russian Karelia, in particular even though the data collection in Russian Karelia has been limited. As far as we are aware no similar studies have been performed earlier. This raises the possibility that nutritional deficiencies and imbalances may be identified. Such information will be useful in the development of dietary guidelines on early infant nutrition childhood diet.
The Main Dissemination Activities in DIABIMMUNE project
When the DIABIMMUNE project started we informed the mass media in Finland. During the whole project-life of DIABIMMUNE the project has caught the interest of both the national media in Finland and the international media. DIABIMMUNE partners have been active in the dissemination work. The Smithsonian Magazine has published the story ‘The Secret Life of Dirt: At the Finnish-Russian border, scientists investigate a medical mystery’. The Smithsonian journalist was travelling with the Study Management Team of University of Helsinki to Petrozavodsk and collecting material for that article. Washington Post has published the article ‘Is there connection between Type 1 diabetes and cleanliness? Finland serves a model’ after the journalist had visited in Helsinki, Finland and interviewed professor Knip, the head of DIABIMMUNE project. The Futuris of EuroNews (www.euronews.com) which presents the leading scientific & technological research projects in Europe, has produced the film ‘Over-hygienic parents could be cause of diabetes’ after visiting in Finland for collecting the material at Jorvi Hospital, Espoo, Finland and at Biomedicum Helsinki where the DIABIMMUNE Core Lab is located. Youris (www.youris.com) has produced the film telling the main story of DIABIMMUNE project ‘Does Too Much Hygiene Cause Diabetes?’. Pan European Networks has published the article ‘Stopped at the Border’. The articles ‘Environmental Biodiversity and Development of Type 1 Diabetes and Other Immune-Mediated Diseases in Children’ as well as ‘The Role of Hygiene in the Development of Type 1 Diabetes’ written by professor Mikael Knip, have been published by the Parliament Magazine.
Several posters have been presented in international, scientific congresses; the poster ‘Differential gene expression of immunologically active molecules between children born in Finland, Estonia and Russian Karelia’ in August 2013 at the 14th International Conference on Systems Biology, Copenhagen, Denmark and the poster ‘Differential gene expression of immunologically active molecules between children born in Finland, Estonia and Russian Karelia’ in June 2013 at the 10th International Workshop on Computational Systems Biology, Tampere, Finland. The poster ‘Early postnatal growth in children with HLA-conferred susceptibility to type 1 diabetes’ has been presented in December 2011 at the Annual meeting of European Society of Paediatric Endocrinology (ESPE) in Leipzig, Germany and the poster ‘Prevalence of early allergic sensitization in children with HLA-conferred susceptibility to type 1 diabetes’ in September 2013 at the Annual meeting of ESPE in Milan, Italy. Petrozavodsk State University and the Ministry of Health and Social Development of Russian Karelia, Russian Federation have conducted active dissemination work in order to change the attitude of the local families towards scientific studies and to motivate them to participate in the DIABIMMUNE project. The advertisements and the interviews of the local Russian DIABIMMUNE partners have been published via the Russian TV channel Rossiya and the local, Russian Karelian TV channel SAMPO. In Petrozavodsk, Russian Karelia the active promotion work has been carried out also by having oral presentations to the local families about T1D and other autoimmune diseases and by distributing flyers and posters about the DIABIMMUNE project to the local kindergartens and the paediatric policlinics.
The following scientific articles have been published for informing the scientific audience: ‘Birth weight in newborn infants with different diabetes-associated HLA genotypes in three neighbouring countries: Finland, Estonia and Russian Karelia’ in the journal Diabetes/Metabolism Research and Reviews, ‘Early postnatal growth in children with HLA-conferred susceptibility to type 1 diabetes’ also in the journal Diabetes/Metabolism Reseach and Reviews. ‘Development of a luciferase-based system for detection of ZnT8 autoantibodies’ is going to be published in Journal of Immunological Methods (in press), ‘Fecal Microbiota Composition Differs Between Children With Beta-Cell Autoimmunity and Those Without’ in the journal of Diabetes, ‘The ‘Hygiene hypothesis’ and the sharp gradient in the incidence of autoimmune and allergic diseases between Russian Karelia and Finland’ in APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, ‘Enterovirus infections in early childhood and the risk of atopic disease - a nested case-control study in Clinical and Experimental Allergy, No association between vitamin D and β-cell autoimmunity in Finnish and Estonian children’ has been accepted for publication in Diabetes/Metabolism Research and Reviews. A series of other scientific articles have been submitted or are in the process to be submitted with novel information on T1D, allergy and hygiene hypothesis.
List of Websites:
The public website address of the project: www.diabimmune.org
The contact details of the scientific coordinator of the DIABIMMUNE Project:
Professor Mikael Knip, University of Helsinki, Finland
Tel. +358 9 471 72701
Email: mikael.knip@helsinki.fi
Preliminary data indicate that there is a strong association between the incidence of immune-mediated diseases and improving standard of living and hygiene. One of the steepest gradients in standard of living worldwide is present at the border between Russian Karelia and Finland with a sevenfold difference in the gross national product, while Estonia represents a country in rapid transition. These three populations comprise a “living laboratory” providing a unique possibility to test the hygiene hypothesis and gene-environmental interactions in the development of immune-mediated diseases. According to the hygiene hypothesis there is an association between the increased incidence and prevalence of immune-mediated diseases and decreased exposure to pathogens. The incidence of T1D is six times lower in Russian Karelia than in Finland, whereas there are very limited differences in the frequency of predisposing and protective HLA (human leukocyte antigen) genotypes in the background population. The aim of the DIABIMMUNE project is to assess the role of the hygiene hypothesis in the development of immune-mediated diseases, T1D in particular, and to define the mechanisms behind the potential protective effect conferred by microbial agents. The objective was to study newborn infants with increased genetic risk to type 1 diabetes and to follow children at the age of 3 and 5 years in Finland, Russian Karelia and Estonia. The children are tested for organ-specific autoantibodies, allergies, infections, gut microflora, and for nutritional factors.
Extensive biobanks comprising genetic samples, serum samples, viral and allercological samples as well as stool samples from study subjects have been established in the DIABIMMUNE project. This material has been used to study the disease process resulting in overt type 1 diabetes or any allergic disease. A strongly protected real-time database including all information collected and all research data generated has been established. Altogether 8863 families gave informed consent to genetic screening of their newborn infant. By the end of February, 2014 altogether 3740 families have attended the first study centre visit with their 3-year old child in Finland, Estonia and Russian Karelia and 7250 follow-up visits for the young children cohort have been performed in the DIABIMMUNE study centres.
Altogether 14.689 HLA samples have been genotypes in the laboratory of University of Turku. Analyses of diabetes-associated autoantibodies [against insulin (IAA), glutamic acid decarboxylase (GADA), insulinoma-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A)] have been performed in 13,335 samples in the autoantibody laboratory, University of Helsinki. The coeliac disease marker antibodies (IgA-TTGA with EliA or/and ELISA, IgG-TTGA, IgG-DGPA, EMA) have been tested in 16,403 blood samples. National Institute for Welfare and Health has received altogether 581 peripheral blood samples from children participating in the birth cohort arm and 341 samples from the cohort of young children. Flow cytometry studies, T-cell suppression tests or phenotyping of regulatory T-cells have been performed from the samples. University of Turku has received 1628 Tenmpus RNA samples from the participants in the birth cohort and 1632 from participants in the young children cohort for RNA isolation and quality control. Four hundred twenty-three samples have been processed for RNA isolation and genomic DNA. The work package of natural history of allergy has produced the knowledge of frequencies of allergic diseases and atopic sensitisation in cohorts of Finnish, Estonian, and Russian Karelian children. These studies have shown that atopic sensitisation and allergic diseases are definitely more common in Finnish children followed by Estonian children, while Russian Karelian children have less sensitisation and allergy. University of Tampere has analysed the frequency of virus infections in the three populations by detecting selected viruses and parasites in stool samples. In addition, selected respiratory viruses were analysed from nasal swab samples. The data in the gut microflora studies analysed by the Academic Hospital Groningen shows that gut colonization of newborn infants is related to the duration and exclusivity of breastfeeding. Especially the colonization with specific bifidobacterial species showed a relation with autoantibody positivity and type 1 diabetes. University of Tampere is responsible for the dietary studies. Altogether 1829 3-day food records have been collected in the birth cohort and in the young children cohort for further analysis.
The DIABIMMUNE project represents translational research with a strong clinical component. The study cohorts comprise children and addresses common immune-mediated diseases, presenting in a majority of the affected cases in childhood, but with a life-long duration as chronic disorders. The study focuses on early events in the development of type 1 diabetes. The DIABIMMUNE project has important socio-economic aspects. The incidence of type 1 diabetes has increased several fold in most industrialised countries over the last 50 years. Accordingly there is a definite need of effective strategies to stop the increase in disease incidence and the ultimate aim would be to turn the trend around, i.e. to effectively decrease the incidence of type 1 diabetes in the developed countries. The data generated so far supports the hygiene hypothesis but it is too early to conclude whether the total microbial load or specific microbes represent the critical culprit.
Project Context and Objectives:
The hygiene hypothesis postulates a relationship between the increasing incidence and prevalence of immune-mediated diseases in modern society and a childhood environment resulting in a decreased pathogen exposure. The hypothesis was initially introduced to explain the increasing frequency of allergy and asthma in developed countries. Recently the hygiene hypothesis has been implicated to contribute to the increasing incidence of autoimmune diseases such as type 1 diabetes (T1D).
The overall objective of the DIABIMMUNE Project has been to test the hygiene hypothesis in T1D and delineate the mechanisms mediating the influence of the standard of hygiene and living on the immune system and leading to a dysregulated immune system predisposing to T1D and allergy.
The specific scientific objectives are
a) to characterise the natural history of beta-cell autoimmunity in three cohorts of young children(Estonia, Finland and Russian Karelia) exposed to contrasting standards of hygiene and living;
b) to identify environmental triggers and determinants of beta-cell autoimmunity;
c) to define gene-environmental interactions in the emergence of signs of beta-cell autoimmunity;
d) to delineate the ontogeny of the immune system in infants and young children living in three contrasting environments by using modern tools of functional genomics;
e) to define the maturation and function of regulatory T cells in infants exposed to contrasting standard of hygiene and living;
f) to analyse the impact of gut microbial colonisation on the ontogeny of the immune system in young infants;
g) to assess the effect of various dietary factors on gut microbial flora and on immune responses induced by acute microbial infections;
h) to define the role of gut microbial flora, acute microbial infections and consumption of foods affecting these microbes in the development of T1D; and
i) to compare the above mentioned variables between children who develop beta-cell autoimmunity and those who present with allergy to identify possible common ethiopathogenetic factors and immunopathogenetic pathways.
The DIABIMMUNE Project has had the following technological objectives:
(i) to establish a biobank comprising genetic samples (DNA and dry blood spots) from newborn infants in Finland, Estonia and Russian Karelia and at least 3500 3-year-old children and genetic samples from 3500 nuclear families (as a minimum a trio including the father, mother and the index cases) from Finland, Estonia and Russian Karelia;
(ii)to screen 6660 newborn infants and 3500 3-year-old children for HLA-conferred susceptibility to T1D and autoimmunity;
(iii)to establish a biobank comprising at least 960 individuals with sequential biological samples taken at intervals of 1-6 months starting from birth. These samples include serum, peripheral blood mononuclear cells, rectal swabs and stools. In addition RNA samples from peripheral blood are collected and stored from a smaller cohort, and breast milk samples are collected from lactating mothers when the index case is 3-month-old;
Project Results:
During the 5 years of the study a lot of data has been collected and the novel knowledge has been generated and continues to be genrated while the data collection is still continuing in Russian Karelia due to the delayed recruitment of the 3-year-old children in the young children cohort. Most analyses are also still continuing.
Extensive biobanks comprising genetic samples from subjects living in three different countries, Finland, Estonia and Russian Karelia, has been established in DIABIMMUNE project. These biobanks can be used to assess the role of genetic elements involved in the development of organ-specific autoimmune and allergic diseases. The project has resulted in a series of other biobanks comprising different biological materials from the subjects taking part in the surveillance part of the project. Such material can be used to study the disease process resulting in overt type 1 diabetes (T1d) or any allergic disease.
A strongly protected real-time database including all information collected and all research data generated has been established starting from the inclusion of the first index case into the study until the last follow-up visit of the study. This database has been utilised for monitoring the progress of the study and for data analysis related to the objectives of the study. The identification of the study participants is based on individual codes in the central database without using personal identifiers. In addition there is in each recruiting study centre a free-standing database combining the individual study code with data identifying the person behind the code.
The recruitment for the birth cohort started in September 2008 and was completed in Finland and Estonia in May 2010 and in Russian Karelia in March 2011. There was a need to extend the recruitment in Russian Karelia due to the lower than expected consent rate to the follow-up among families with an HLA-eligible baby. By the end of February, 2014 altogether 8863 families gave informed consent to genetic screening of their newborn infant. Almost 19% (1653; 18.6 %) of the infants screened were observed to carry HLA-conferred susceptibility to autoimmunity, which is very close to the expected proportion of 18%. Eight hundred twenty-nine families have decided to join the follow-up study of HLA-eligible subjects in the birth cohort. Altogether 4003 follow-up visits have been performed for the birth cohort children in the DIABIMMUNE study centres. The recruitment for the young children cohort started in the fall 2009 in Espoo, Finland and in Tartu, Estonia. Petrozavodsk, Russian Karelia started in October 2011. By the end of February, 2014 3740 families have attended the first study centre visit with their 3-year-old child. The recruitment was closed in June 2011 in Tartu with 1681 families participating and in July 2011 in Espoo with 1575 families. In Petrozavodsk, Russian Karelia the recruitment for the young children cohort was finalized in March 2014 with 484 families. Altogether 7250 follow-up visits have been performed for the young children cohort in the DIABIMMUNE study centres. The last follow-up visits were conducted in Espoo, Finland in June 2013 and in Tartu, Estonia in September 2013. The follow-up in Russian Karelia is still going on.
The HLA laboratory in Turku, Finland, has worked effectively from September, 2008. Altogether 14,689 HLA samples have been analysed by the end of February 2014.
The analyses of diabetes-associated autoantibodies [against insulin (IAA), glutamic acid decarboxylase (GADA), insulinoma-2 antigen (IA-2A), and zinc transporter 8 (ZnT8A)] have been performed in the autoantibody laboratory, University of Helsinki. In the young children cohort, six Finnish and four Estonian children have so far presented with clinical T1D. All affected children have tested positive for multiple autoantibodies during the follow-up. Progression to T1D was twice as common in Finnish children as in Estonian children, even though, due to the low numbers, the difference was not statistically significant. The fact that no case of T1D hase been identified in Russian children thus far is within the expected incidence range characteristic of the paediatric population of Russian Karelia.
The coeliac disease marker antibodies (IgA-TTGA with EliA or/and ELISA, IgG-TTGA, IgG-DGPA, EMA) have been tested in 16,403 blood samples. The IgA-TTGA levels ranged from 0 to 4262 U/ml, IgG-TTGA levels between 0 and 113 U/ml, and IgG-DGPA levels between 0 and 245 U/ml. There was a suspicion of IgA deficiency in 19 cases, but all serum samples were IgG-TTGA and IgG-DGPA negative. The serum samples from the birth cohort children comprised a umbilical cord blood sample sand serum samples taken at 3, 6, 12, 18, 24 and 36 months of age. Altogether nine children had developed IgA-TTGA during the 3-year follow up. In all nine children coeliac disease was confirmed by histological examination of small bowel biopsies. Seven (1.8 %) children out of 387 children from Espoo and two (0.61 %) out of 330 children from Tartu enrolled in follow-up tested positive for IgG-TTGA in cord blood.
The coeliac disease marker antibodies in the serum samples from the children aged 3, 4 and 5 years (n=6868) in the young children cohort and from their siblings (n=755) and parents (n=143) were also analysed. Combining the results in the index childrem we discovered that 21 (1.3 %) children in the original Espoo (n=1575) and 14 (0.8 %) children in the original Tartu cohort (n=1681) have tested positive for IgA-TTGA once or several times by 5 years of age. In the Petrozavodsk cohort (n=310) two children (0.65%) turned out to have coeliac disease associated antibodies. Based on these results the children were invited to gastroduodenoscopy and small bowel biopsy. Altogether, 22 children were biopsied and coeliac disease was histologically confirmed in 19 (13 from Espoo and six from Tartu). In three children coeliac disease was not detected in spite of the presence of IgA-TTGA. Among these children one had shown repeatedly high IgA-TTGA values, one had moderately elevated antibody levels and one tested only once weakly positive.
The National Institute for Welfare and Health has received altogether 581 peripheral blood mononuclear cell samples from children participating in the birth cohort arm and 341 samples from the cohort of young children by the end of February 2014. Flow cytometry studies, T-cell suppression tests or phenotyping of regulatory T-cells have been performed from the samples. In the T-cell studies has been compared The number and function of regulatory T cells have been compared between Estonian and Finnish children in the T-cell studies. No fresh blood samples have been received from Russian Karelia due to problems with the approval of the transportation of biological samples from Russian Federation and the impossibility to arrange the sample delivery to Helsinki, Finland within 24 hours after the obtaining fresh T-cell samples in Petrozavodsk.
The observation of increased numbers of highly activated Treg cells with high expression of FOXP3 at 3 months of age in Estonian children suggest that regulatory mechanisms are more active after birth in Estonian than Finnish children. This could be due to differences in the microbial load between the two countries and the analyses of the association of gut microbiota and Treg cells, which are on-going, may reveal bacterial strains or species with potential therapeutic possibilities for the activation of T-regs. The findings of regulatory T-cell studies show that the emergence of a subpopulation of highly activated Tregs is seen when the child’s immune system matures during the first 3 years of life
University of Turku has received 1628 follow-up Tempus RNA samples from the birth cohort children and 1632 samples from the young children cohort by the end of February 2014 for RNA isolation and quality control. Four hundred twenty-three samples have been processed for RNA isolation and genomic DNA. Data generated for functional genomics has produced novel information on the effect on the standard of hygiene on the expression of critical molecules affecting the immune system. The transcriptomics measurements of the cord blood samples collected at the time of birth clearly indicated, that the children born in Petrozavodsk, Russian Karelia, deviate from children born in Espoo, Finland or Tarto, Estonia. The results suggest that newborn infants in Russian Karelia have been exposed to TLR ligands in utero, and that the subsequent immune response is developmentally more mature. Therefore, lack of in utero immunomodulation may influence the predisposition towards immune mediated diseases in the Westernized world. These data will lead to further studies focusing on the in utero period in the regulation of maturation and activation of the immune system. The results have been communicated to the scientific community through poster presentations in two international scientific meetings, and a manuscript describing the results has been submitted for publication in an international peer-reviewed scientific journal. In addition, further investigations and analyses ongoing in the Work package 6 will focus on investigating the transcriptomics changes occuring A) during immune system maturation (3 months -3 years), and B) during appearance of T1D-associated autoantibodies (Birth cohort and Young Children Cohort). The results will be published as separate articles im scientific journals.
The work package of natural history of allergy has produced the knowledge of frequencies of allergic diseases and sensitisation in cohorts of Finnish, Estonian, and Russian Karelian children born between 2005 and 2011. The allergy studies have shown clear differences between the Estonian, Finnish and Russian Karelian children. The allergic sensitisation is less prevalent in Estonian and Russian Karelian children in the birth cohort than in the Finnish peers. Similar results were observed seen among 3-5-year-old children in Finland and in Estonia.
University of Tampere has continued to develop new technologies for screening of microbes from stool, serum and nasal swab samples and carried out analyses of samples collected. The frequency of virus infections was analysed in the three populations by detecting selected viruses and parasites in stool samples. In addition, selected respiratory viruses were analysed from nasal swab samples. Enteroviruses were found to be most frequent in young children in Russian Karelia, followed by Estonian children, while they were detected more rarely in Finnish children. However, norovirus and parechovirus infections did not differ between the countries and rhinovirus infections were most frequent in Finnish and Estonian children and less frequent in Russian Karelian children. The data generated in the work package of microbial infections indicates that virus infections have an effect of immune regulation and development of IgE-mediated sensitization. This offers possibilites to use these viruses or their immunologically acitive structural components in the development of new treatments for allergic diseases.
The goal of Academic Hospital Groningen has been to delineate the gut microbial colonization in infants exposed to contrasting standards of hygiene and to investigate which specific intestinal microbes are involved in the development of T1D and other immune-mediated diseases in 3 and 5-year old children. The data generated by the gut microflora studies show that the intestinal colonization of newborn infants is related to the duration and exclusivity of breastfeeding. Especially the colonization with specific bifidobacterial species showed a relation with autoantibody positivity and T1D. The colonization after weaning showed that higher numbers of the Bacteroides genus, a lower abundance of species from Clostridium cluster IV, a lower abundance of particular butyrate producers from Clostridium cluster XIVa and fewer Prevotella were present in Finland. Finnish children had a different microbiota than the Estonian children who are characterised by a flora that is now generally assumed to be more protective.
The work plan included more detailed HLA genotyping and genotyping for major T1D associated non-HLA genes including INS, PTPN22 and CTLA4 in the subgroups of children developing beta-cell specific autoimmunity. This has not been yet performed as the final autoantibody results have just become available and the control group for children with autoimmunity has not yet been selected.
University of Tampere is also responsible for the dietary studies. Altogether 1829 3-day food records have been collected in the birth cohort and in the young children cohort for further analysis. Among children 0-36 months of age, 3-day food records have been collected at the 6 and 12-month visits and among children 3-5 years of age at the 4 and 5-year visits. The food database is completed for all the foods used in the DIABIMMUNE study and the food data entry of the food records is also completed in Finland and in Estonia. In Russia, the collection of food records has been challenging, and the collection continues. The analyses of the dietary data are continuing.
DIABIMMUNE Project has not received all its milestones due to the difficulties and the challenges with the recruitment and the follow-up in Petrozavodsk, Russian Karelia. The follow-up visits of the young children cohort are continuing to be performed in Russian Karelia until the end of the year 2015. The data analyses are also continuing due to the delay and the challenges in the recruitment and the follow-up of Russian Karelian children. The continuation of the study has been made possible by the separate funding received from Academy of Finland, University of Helsinki and Sigrid Juselius Foundation. The DIABIMMUNE study has generated data that can be utilized as background information in the part of the INTERCONNECT project focusing on type 1 diabetes.
Potential Impact:
The study setting has provided a unique opportunity to define the impact of contrasting standards of hygiene and living on the appearance of signs of beta-cell autoimmunity and allergy in young children. The project has generated and is still generating novel knowledge that can be exploited for developing preventive strategies that are able to break the present alarming trend of increasing incidences and prevalences of type 1 diabetes seen in most EU countries. Similar contrasts in standards of hygiene and living can hardly be found anywhere else in Europe or even globally. Finland represents in relative terms a highly developed country, Russian Karelia a so far poorly developed country and Estonia a country in rapid transition from poverty to relative prosperity. The adjacent location of the three countries standardises most geophysical factors, such as temperature, rain, and hours of sunshine and these factors cannot explain the differences observed e.g. in the incidence of type 1 diabetes (T1D) between Finland and Russian Karelia in our previous study. In addition Finns, Karelians and Estonians are genetically related providing an opportunity to assess the direct impact of environmental factors. In Russian Karelia Russians comprise the majority of the population, but the proportion of Karelians is about 12%. In Estonia approximately 26% of the population is of Russian origin making it possible to perform comparative analyses between children of Estonian or Russian origin, both groups born in Estonia.
DIABIMMUNE project represents translational research with a strong clinical component. The study cohorts comprise children and addresses common immune-mediated diseases, presenting in a majority of the affected cases in childhood, but with a life-long duration as chronic disorders. The proposal focuses on early events in the development of T1D by studying children with HLA-conferred disease susceptibility from birth up to the age of 3 years. In addition a genetically unselected, more extensive cohort including 3-year-old children has been recruited and studied. The latter cohort was re-examined 2 years later to generate additional information on the natural history of preclinical T1D and allergy. A multidisciplinary approach was applied as the consortium comprises diabetologists, allergy specialists, virologists, immunologists, experts on infectious diseases, and nutritional scientists. The longer-term goal of the project is to develop intervention strategies mimicking the mechanisms identified behind the protective effect conferred by the Russian-Karelian environment against T1D and other immune-mediated diseases. If the hygiene hypothesis is confirmed, oral administration of safe microbial products (live vaccines and probiotic-type bacterial products), provides a tempting option for a future intervention trial in populations with a high incidence of T1D and other immune-mediated diseases. Microbial products including live vaccines and bacterial components have already been licensed for medical use in Europe.
The DIABIMMUNE project has important socio-economic aspects. The incidence of T1D has increased several fold in most industrialised countries over the last 50 years. Accordingly there is a definite need of effective strategies to stop the increase in disease incidence and the ultimate aim would be to turn the trend around, i.e. to effectively decrease the incidence of T1D in the developed countries. Such strategies require, however, novel knowledge of those factors, which have contributed to the increase in incidence rate of T1D and other autoimmune diseases in Western countries after World War II. The DIABIMMUNE project provides a unique opportunity to generate such new knowledge, since the study has made it possible to compare the maturation of the immune system in young children representing populations with conspicuous contrasts in their standard of hygiene and living. Simultaneously it will be possible to time the autoimmune and allergic process and thereby facilitate the identification of host-related and environmental factors functioning as triggers of the disease process and as determinants of the progression from the initiation of the process to full-blown clinical disease.
T1D is an immune-mediated disease where the destruction of the insulin-producing beta cells is triggered by some so far unidentified exogenous factor(s) in an individual with increased genetic disease susceptibility. The diagnosis of clinical diabetes is preceded by an asymptomatic preclinical period, which may last for months and years. The shortest prediabetic process that we have observed in prospective studies based on the occurrence of diabetes-associated autoantibodies in the peripheral circulation lasted for less than 3 months and the longest for more than 20 years. Currently T1D is an incurable chronic disease. Despite progress in insulin therapy and other aspects of diabetes care over the last 30 years, only a small minority of the affected patients are able to maintain good metabolic control. As a consequence the disease may lead to a series of complications, such as retinopathy, nephropathy and neuropathy resulting in decreased quality of life and reduced life expectancy. From the social point of view T1D is associated with substantial costs. It has been estimated that presentation of T1D in childhood results in Finland in additional lifetime costs amounting to about 1 M€.
Allergic diseases are the most common group of chronic disorders in children and adolescents in the developed countries. The incidence and prevalence of such diseases have increased substantially after World War II in all EU countries. The European Allergy White Paper from 1997 states that allergic diseases have reached epidemic proportions and recognises that these diseases are a public health problem in Europe. The paper also asks for effective preventive measures and emphasises that prevention requires more precise identification of atopic individuals and risk factors in the aetiology of atopic diseases. One aim of the DIABIMMUNE project has been the identification of such risk factors and possible protective factors that can be utilised in the development of effective preventive strategies.
Preventive aspects
The results of this project can potentially be exploited to establish strategies aimed at delaying or preventing progression to clinical T1D and at decreasing the incidence and prevalence of allergy in developed countries, such as the EU countries. By studying the natural history of beta-cell autoimmunity in three countries with a strong gradient in the rate of T1D we have learned more about at which age the first autoantibodies appear and about the rate of progression to clinical disease. Such information can be utilised to assess the window open for primary prevention of T1D. In addition it has provided an opportunity to identify exogenous triggers of beta-cell autoimmunity. The study also facilitates the identification of exogenous factors affecting the progression to clinical disease. The identification of such factors might open up new avenues for preventive measures.
Observation of the natural history of allergy provides information on at which age the immune system is diverted into reactivity favouring allergic responses. This knowledge defines the optimal window for intervention aimed at preventing such diversions. The study setting has also provided opportunities to identify host-related and environmental factors and their mutual interactions triggering allergy and factors potentiating the development of asthma and other allergic diseases. Also factors protecting against allergic diseases might be identified.
Scientific and health-related aspects
It has been shown previously that HLA-conferred susceptibility to T1Dis a strong determinant of the appearance of signs of beta-cell autoimmunity in young children. The DIABIMMUNE project has facilitated the analysis of gene-environment interactions in the emergence of signs of beta-cell autoimmunity and allergy by introducing more heterogeneity both in relation to genetic and environmental factors compared to a birth cohort study implemented in a single country.
By comparing the expression profile of immunologically active molecules with 3-6-month intervals during the first year of life starting from birth between infants living in the three countries taking part in the study it is possible to define the impact of the contrasting standard of hygiene and living on the expression profiles allowing the identification of changes over time. Such expression profiles may generate novel hypotheses that might be further explored based on the various forms of biological material collected from the study subjects.
The study protocol facilitates the assessment of the impact of dietary factors on the maturation of the immune system. This generates novel knowledge that may be used to optimise both early infant nutrition and subsequent diet to support the establishment of normal immune responses. The study design has also allowed a comparison of the colonisation of the gastrointestinal tract in infancy between subjects from the three countries and the analysis of the effect of dietary factors in this context. The interactions between gut microbial flora, acute microbial infections and dietary factors in the development of organ-specific autoimmunity and allergy can be analysed in a versatile way due to the substantial variation in these factors expected to be seen between subjects from the three countries.
We have studied early infant nutrition and the diet in young children within the framework of the current project. This provides novel information on the diet of infants and young children in Estonia and in Russian Karelia, in particular even though the data collection in Russian Karelia has been limited. As far as we are aware no similar studies have been performed earlier. This raises the possibility that nutritional deficiencies and imbalances may be identified. Such information will be useful in the development of dietary guidelines on early infant nutrition childhood diet.
The Main Dissemination Activities in DIABIMMUNE project
When the DIABIMMUNE project started we informed the mass media in Finland. During the whole project-life of DIABIMMUNE the project has caught the interest of both the national media in Finland and the international media. DIABIMMUNE partners have been active in the dissemination work. The Smithsonian Magazine has published the story ‘The Secret Life of Dirt: At the Finnish-Russian border, scientists investigate a medical mystery’. The Smithsonian journalist was travelling with the Study Management Team of University of Helsinki to Petrozavodsk and collecting material for that article. Washington Post has published the article ‘Is there connection between Type 1 diabetes and cleanliness? Finland serves a model’ after the journalist had visited in Helsinki, Finland and interviewed professor Knip, the head of DIABIMMUNE project. The Futuris of EuroNews (www.euronews.com) which presents the leading scientific & technological research projects in Europe, has produced the film ‘Over-hygienic parents could be cause of diabetes’ after visiting in Finland for collecting the material at Jorvi Hospital, Espoo, Finland and at Biomedicum Helsinki where the DIABIMMUNE Core Lab is located. Youris (www.youris.com) has produced the film telling the main story of DIABIMMUNE project ‘Does Too Much Hygiene Cause Diabetes?’. Pan European Networks has published the article ‘Stopped at the Border’. The articles ‘Environmental Biodiversity and Development of Type 1 Diabetes and Other Immune-Mediated Diseases in Children’ as well as ‘The Role of Hygiene in the Development of Type 1 Diabetes’ written by professor Mikael Knip, have been published by the Parliament Magazine.
Several posters have been presented in international, scientific congresses; the poster ‘Differential gene expression of immunologically active molecules between children born in Finland, Estonia and Russian Karelia’ in August 2013 at the 14th International Conference on Systems Biology, Copenhagen, Denmark and the poster ‘Differential gene expression of immunologically active molecules between children born in Finland, Estonia and Russian Karelia’ in June 2013 at the 10th International Workshop on Computational Systems Biology, Tampere, Finland. The poster ‘Early postnatal growth in children with HLA-conferred susceptibility to type 1 diabetes’ has been presented in December 2011 at the Annual meeting of European Society of Paediatric Endocrinology (ESPE) in Leipzig, Germany and the poster ‘Prevalence of early allergic sensitization in children with HLA-conferred susceptibility to type 1 diabetes’ in September 2013 at the Annual meeting of ESPE in Milan, Italy. Petrozavodsk State University and the Ministry of Health and Social Development of Russian Karelia, Russian Federation have conducted active dissemination work in order to change the attitude of the local families towards scientific studies and to motivate them to participate in the DIABIMMUNE project. The advertisements and the interviews of the local Russian DIABIMMUNE partners have been published via the Russian TV channel Rossiya and the local, Russian Karelian TV channel SAMPO. In Petrozavodsk, Russian Karelia the active promotion work has been carried out also by having oral presentations to the local families about T1D and other autoimmune diseases and by distributing flyers and posters about the DIABIMMUNE project to the local kindergartens and the paediatric policlinics.
The following scientific articles have been published for informing the scientific audience: ‘Birth weight in newborn infants with different diabetes-associated HLA genotypes in three neighbouring countries: Finland, Estonia and Russian Karelia’ in the journal Diabetes/Metabolism Research and Reviews, ‘Early postnatal growth in children with HLA-conferred susceptibility to type 1 diabetes’ also in the journal Diabetes/Metabolism Reseach and Reviews. ‘Development of a luciferase-based system for detection of ZnT8 autoantibodies’ is going to be published in Journal of Immunological Methods (in press), ‘Fecal Microbiota Composition Differs Between Children With Beta-Cell Autoimmunity and Those Without’ in the journal of Diabetes, ‘The ‘Hygiene hypothesis’ and the sharp gradient in the incidence of autoimmune and allergic diseases between Russian Karelia and Finland’ in APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, ‘Enterovirus infections in early childhood and the risk of atopic disease - a nested case-control study in Clinical and Experimental Allergy, No association between vitamin D and β-cell autoimmunity in Finnish and Estonian children’ has been accepted for publication in Diabetes/Metabolism Research and Reviews. A series of other scientific articles have been submitted or are in the process to be submitted with novel information on T1D, allergy and hygiene hypothesis.
List of Websites:
The public website address of the project: www.diabimmune.org
The contact details of the scientific coordinator of the DIABIMMUNE Project:
Professor Mikael Knip, University of Helsinki, Finland
Tel. +358 9 471 72701
Email: mikael.knip@helsinki.fi