**1. Introduction**

336 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

[69] Maahs, D.M., et al., *Epidemiology of type 1 diabetes.* Endocrinol Metab Clin North Am,

Type 1 Diabetes (T1D) is one of the most common chronic diseases with childhood onset, and the disease has increased two to threefold over the past half century by yet unknown means. Recently it was showed that if the present trend continues, the prevalence of cases younger than 5 years of age will rise by 70% within year 2020 (Patterson et al., 2009).

#### **1.1 Background and status of knowledge**

Type 1 Diabetes (T1D) is a T-cell mediated autoimmune disease that develops in genetically susceptible individuals whose immune system destroys the majority of insulin-secreting βcells in pancreatic islets (Eizirik et al., 2009). The incidence of T1D has increased more than two- to threefold over the past half century, the most striking example being Finland where it has risen from 12 to 63/100,000 (Knip & Siljander, 2008; Patterson et al., 2009). This increase in incidence has not been paralleled by an increase in the frequency of major risk genes, including HLA class II, insulin, PTPN22, CTLA-4 and IL2RA (Barrett et al., 2009). Indeed, the prevalence of the classical HLA class II genes, which account for approximately 40% of genetic risk, appears to be decreasing (Gillespie et al., 2004; Fourlanos et al., 2008). There are now more than 40 risk loci associated with T1D with the majority of non-HLA genes displaying odds ratio <1.2. (Barrett et al., 2009). Moreover, most individuals who possess T1D risk genes do not develop the disease. Importantly, the concordance rate among monozygotic twins ranges from as low as 25 to 65% (Redendo et al., 1999, 2008; Hyttinen et al., 2003) and is approximately 6% in siblings. A common explanation has been that changes in environment must contribute to the increase in the disease. In particular, environmental exposures to dietary antigens and microbes have been implicated (Knip et al., 2005; Lefebvre et al., 2006). However, no single pathogenic environmental agent has been identified that explain all cases. In all likelihood, T1D develops by various combinations of pathways in response to commonly encountered environmental exposures.

#### **1.2 Nutritional related factors and type 1 diabetes risk**

The Norwegian Institute of Public Health is currently running two large prospective cohort studies; "Environmental Triggers of Type 1 Diabetes" (MIDIA) (www.fhi.no/midia) and "The Norwegian Mother and Child Cohort Study" (MoBa) (www.fhi.no/morogbarn). In MIDIA we will be able to study the impact of the dietary intake in children as well as

Genetic Testing of Newborns for Type 1 Diabetes Susceptibility – The MIDIA Study 339

(Virtanen et al., 2006). While a recent study found that higher maternal intake of potatoes in the last trimester of pregnancy was associated with delayed onset of autoimmunity in the offspring (Lamb et al., 2008). Inconsistencies between the studies indicate that additional studies are required, including resolving the question of what aspects of cereals or other

A Norwegian study found intake of cod liver oil by the mothers during pregnancy or possibly by the child during the first year of life to be associated with lower risk of T1D in the child (Stene et al., 2000), but a subsequent larger study indicated that the child's intake was most important (Stene & Joner, 2003). Other vitamin D supplements were not associated in the Norwegian studies, pointing towards a possible effect of long-chain omega-3 fatty acids. Such fatty acids (e.g. EPA, DHA) have anti-inflammatory effects and potentially preventive effects for T1D (Chase et al., 1979). Results from a case-control study indicated, however, that vitamin D supplementation in early childhood could protect against T1D , and this has also been supported both from an European collaborative study (EURODIAB, 1999) as well as in a prospective study of children born in 1965 in Finland (Hyppönen et al., 2001). The longitudinal, observational study, the Diabetes Study in the Young (DAISY), conducted in Denver, Colorado, between January 1994 and November 2006, suggested that higher consumption of total omega-3 fatty acids, which was reported by a food frequency questionnaire, was associated with a lower risk of autoimmunity in children at increased genetic risk for type 1 diabetes. This association was further sustained by the observation of a higher proportion of omega-3 fatty acids found in the erythrocyte membranes in a subset of the children. Given that fish are a source of both omega-3 fatty acids and vitamin D, vitamin D was initially included in the analysis, but no association was found (Norris et al., 2007). Neither was it support for an effect of marine omega-3 fatty acids analysed separately. Pilot data from the MIDIA study do, however, indicate a protective effect against progression from autoimmunity to development of T1D (unpublished data). Although epidemiological studies can suggest possible associations, randomized clinical trials are necessary to prove a cause and effect. The pilot trial Nutritional Intervention to Prevent (NIP) T1D among babies with high genetic risk was therefore recently initiated

Hypothesising that antioxidants may protect against destruction of-cells, Finnish researchers measured serum -tocopherol concentration in frozen sera from 19 cases who developed T1D and in about 60 individually matched controls from a prospective cohort of individuals aged above 20 years (Knekt et al., 1999). Higher -tocopherol was associated with a significantly lower risk of T1D. Another study from Finland, attempted to replicate this finding in siblings of persons with T1D, and found partial support, although the results were not significant (Uusitalo et al., 2005). Recently both concentration of - and tocopherol were studied in the Type 1 Diabetes Prediction and Prevention project (DIPP). Although it seemed unlikely that high concentration of - or -tocopherol protect against advanced cell autoimmunity in young children, there was a suggestive protective effect of high levels of-tocopherol at the age of 1 year on development of autoimmunity, which

solid food items which are involved.

(Chase et al., 2009).

needs to be replicated (Uusitalo et al., 2008).

**1.2.5 Vitamin E** 

**1.2.4 Cod liver oil, vitamin D and omega-3 fatty acids** 

mothers during the breast-feeding period and in MoBa we will be able to study the dietary intake of the mother during pregnancy for development of T1D in the child. These two studies will be linked to allow several approaches to be tested in the role of early diet and development of T1D. In the MIDIA study newborns have been identified by testing for the high-risk genotype (DRB1\*03-DQA1\*05-DQB1\*02/ DRB1\*04:01-DQA1\*03-DQB1\*03:02) in the HLA-system (Cinek et al., 2000). The MIDIA study is unique compared to the few other ongoing worldwide cohorts because of pregnancy data from the Norwegian Mother and Child Cohort Study (MoBa). Information from questionnaires, public records as well as blood samples from mothers (twice during pregnancy) and children (cord blood) have been collected from 107,000 pregnancies (Magnus et al., 2006; Rønningen et al., 2006). 50% of the children participating in MIDIA have a mother who also participates in MoBa, and consent has been given for linking information from the two studies and using biological specimens (Stene et al., 2007).

#### **1.2.1 Foetal exposure and early life exposure to nutritional factors**

Many nutritional factors may operate in uteri (measured as the mother's exposure during pregnancy), and also during postnatal life, and the status of the child is often influenced both by the maternal intake during pregnancy and postnatal exposures. Because the most relevant timing of exposure and possible induction times are unknown for T1D, an approach addressing both intrauterine and postnatal exposure to hypothetical risk factors or protective factors is most sensible.

#### **1.2.2 Breast-feeding and cow's milk**

Several epidemiological studies indicate that the risk of T1D is lower in children that have been breast-fed compared to children given breast-milk substitute produced from cow's milk (Norris & Scott., 1996), and recent data also indicate that for avoiding early autoimmunity the duration of breastfeeding is of importance (Rosenbauer et al., 2008). But most case-control studies suffer from potential recall bias, and prospective studies up to now have been few and very small. Case-control studies have also found associations between cow's milk antibodies and T1D see e.g. (Sarugeri et al., 1999; Monetini et al., 2002), but some form of reverse causality cannot be excluded as alternative explanations for the association described in these studies. Although an early study indicated a role of so-called molecular mimicry between a protein in cow's milk and a -cell antigen (Karjalainen et al., 1992), it was subsequently refuted (Rønningen et al., 1998). Multiple other biologically plausible mechanisms have also been proposed for the possible relation between short duration of breast-feeding or early introduction of cow's milk.

#### **1.2.3 Introduction of solid food**

Studies indicate that the time point for introduction of solid food, especially with regard to cereal products, may have an influence on the development of autoimmunity (Norris et al., 2003; Ziegler et al., 2003). Early exposure to cereals is against generally accepted recommendations on infant nutrition in all developed countries and occurs infrequently. For example, Scandinavian babies are rarely exposed to cereals before the age of 4 months. A prospective analysis of data from the Finnish Diabetes Prediction and Prevention (DIPP) study showed no relation between early or late introduction of cereals and emergence of advanced β-cell autoimmunity. Another study from Finland suggests that an early introduction of fruit, berries and roots associated independently with β-cell autoimmunity (Virtanen et al., 2006). While a recent study found that higher maternal intake of potatoes in the last trimester of pregnancy was associated with delayed onset of autoimmunity in the offspring (Lamb et al., 2008). Inconsistencies between the studies indicate that additional studies are required, including resolving the question of what aspects of cereals or other solid food items which are involved.

#### **1.2.4 Cod liver oil, vitamin D and omega-3 fatty acids**

A Norwegian study found intake of cod liver oil by the mothers during pregnancy or possibly by the child during the first year of life to be associated with lower risk of T1D in the child (Stene et al., 2000), but a subsequent larger study indicated that the child's intake was most important (Stene & Joner, 2003). Other vitamin D supplements were not associated in the Norwegian studies, pointing towards a possible effect of long-chain omega-3 fatty acids. Such fatty acids (e.g. EPA, DHA) have anti-inflammatory effects and potentially preventive effects for T1D (Chase et al., 1979). Results from a case-control study indicated, however, that vitamin D supplementation in early childhood could protect against T1D , and this has also been supported both from an European collaborative study (EURODIAB, 1999) as well as in a prospective study of children born in 1965 in Finland (Hyppönen et al., 2001). The longitudinal, observational study, the Diabetes Study in the Young (DAISY), conducted in Denver, Colorado, between January 1994 and November 2006, suggested that higher consumption of total omega-3 fatty acids, which was reported by a food frequency questionnaire, was associated with a lower risk of autoimmunity in children at increased genetic risk for type 1 diabetes. This association was further sustained by the observation of a higher proportion of omega-3 fatty acids found in the erythrocyte membranes in a subset of the children. Given that fish are a source of both omega-3 fatty acids and vitamin D, vitamin D was initially included in the analysis, but no association was found (Norris et al., 2007). Neither was it support for an effect of marine omega-3 fatty acids analysed separately. Pilot data from the MIDIA study do, however, indicate a protective effect against progression from autoimmunity to development of T1D (unpublished data). Although epidemiological studies can suggest possible associations, randomized clinical trials are necessary to prove a cause and effect. The pilot trial Nutritional Intervention to Prevent (NIP) T1D among babies with high genetic risk was therefore recently initiated (Chase et al., 2009).

#### **1.2.5 Vitamin E**

338 Type 1 Diabetes – Complications, Pathogenesis, and Alternative Treatments

mothers during the breast-feeding period and in MoBa we will be able to study the dietary intake of the mother during pregnancy for development of T1D in the child. These two studies will be linked to allow several approaches to be tested in the role of early diet and development of T1D. In the MIDIA study newborns have been identified by testing for the high-risk genotype (DRB1\*03-DQA1\*05-DQB1\*02/ DRB1\*04:01-DQA1\*03-DQB1\*03:02) in the HLA-system (Cinek et al., 2000). The MIDIA study is unique compared to the few other ongoing worldwide cohorts because of pregnancy data from the Norwegian Mother and Child Cohort Study (MoBa). Information from questionnaires, public records as well as blood samples from mothers (twice during pregnancy) and children (cord blood) have been collected from 107,000 pregnancies (Magnus et al., 2006; Rønningen et al., 2006). 50% of the children participating in MIDIA have a mother who also participates in MoBa, and consent has been given for linking information from the two studies and using biological specimens

Many nutritional factors may operate in uteri (measured as the mother's exposure during pregnancy), and also during postnatal life, and the status of the child is often influenced both by the maternal intake during pregnancy and postnatal exposures. Because the most relevant timing of exposure and possible induction times are unknown for T1D, an approach addressing both intrauterine and postnatal exposure to hypothetical risk factors or

Several epidemiological studies indicate that the risk of T1D is lower in children that have been breast-fed compared to children given breast-milk substitute produced from cow's milk (Norris & Scott., 1996), and recent data also indicate that for avoiding early autoimmunity the duration of breastfeeding is of importance (Rosenbauer et al., 2008). But most case-control studies suffer from potential recall bias, and prospective studies up to now have been few and very small. Case-control studies have also found associations between cow's milk antibodies and T1D see e.g. (Sarugeri et al., 1999; Monetini et al., 2002), but some form of reverse causality cannot be excluded as alternative explanations for the association described in these studies. Although an early study indicated a role of so-called molecular mimicry between a protein in cow's milk and a -cell antigen (Karjalainen et al., 1992), it was subsequently refuted (Rønningen et al., 1998). Multiple other biologically plausible mechanisms have also been proposed for the possible relation between short

Studies indicate that the time point for introduction of solid food, especially with regard to cereal products, may have an influence on the development of autoimmunity (Norris et al., 2003; Ziegler et al., 2003). Early exposure to cereals is against generally accepted recommendations on infant nutrition in all developed countries and occurs infrequently. For example, Scandinavian babies are rarely exposed to cereals before the age of 4 months. A prospective analysis of data from the Finnish Diabetes Prediction and Prevention (DIPP) study showed no relation between early or late introduction of cereals and emergence of advanced β-cell autoimmunity. Another study from Finland suggests that an early introduction of fruit, berries and roots associated independently with β-cell autoimmunity

**1.2.1 Foetal exposure and early life exposure to nutritional factors** 

duration of breast-feeding or early introduction of cow's milk.

(Stene et al., 2007).

protective factors is most sensible.

**1.2.3 Introduction of solid food** 

**1.2.2 Breast-feeding and cow's milk** 

Hypothesising that antioxidants may protect against destruction of-cells, Finnish researchers measured serum -tocopherol concentration in frozen sera from 19 cases who developed T1D and in about 60 individually matched controls from a prospective cohort of individuals aged above 20 years (Knekt et al., 1999). Higher -tocopherol was associated with a significantly lower risk of T1D. Another study from Finland, attempted to replicate this finding in siblings of persons with T1D, and found partial support, although the results were not significant (Uusitalo et al., 2005). Recently both concentration of - and tocopherol were studied in the Type 1 Diabetes Prediction and Prevention project (DIPP). Although it seemed unlikely that high concentration of - or -tocopherol protect against advanced cell autoimmunity in young children, there was a suggestive protective effect of high levels of-tocopherol at the age of 1 year on development of autoimmunity, which needs to be replicated (Uusitalo et al., 2008).

Genetic Testing of Newborns for Type 1 Diabetes Susceptibility – The MIDIA Study 341

(Blaser, 1998; Malaty, 1994). There are fewer studies in man suggesting protective effect of childhood infections against T1D (Sepp et al., 1997; Samulsson & Ludvigsson, 2003;

Since immunomodulatory effects of parasites have been reported (Samulsson & Ludvigsson, 2003), and there is evidence that infections protect against the development of allergic disorders, parasites become obvious and major candidates for the hygiene hypothesis. In 1947 it was reported that 40-60% of European children were positive for helminths (Horman et al., 2004), while in recent years only 5-23% are found positive (Strachan, 1989; Jones et al., 2000; Yazdanbakhsh et al., 2002; Cooke, 2009; Bach, 2002; Honeyman, 2005; van der Werf et al., 2007; Gibbon et al., 1997; Parslow et al., 2001; Pundziute-Lycka et al., 2003; Round & Mazmanian, 2009). The most prevalent of the helminths is Enterobius Vermicularis (pinworm) which is usually asymptomatic and each bout is self-limiting since the worms cannot reproduce within the gut. Most common of the water borne parasites are Cryptosporium and Giardia. While the genus Giardia comprises

six species, more than 20 variants of Cryptosporium are known (Nygard et al., 2003).

Another interesting possibility is that the age at infection makes a difference in the pathology. In a similar fashion, it has been shown that colonization of the gut and intestines in early infancy by bacteria plays a role in the development of the adaptive immune system and structural development of the gut (Strachan, 1989). It is well known that due to improved hygiene some viral infections that would normally occur in early life are encountered for the first time at a later stage. For example, mononucleosis is associated with late infection of Epstein-Barr virus (EBV) (Pohl, 2009). Mononucleosis is rare in third world countries. Similarly, hepatitis A and B are less likely to cause disease if exposed to at an early age, and chickenpox (caused by varicella-zoster virus) is more severe in adults. Apparently, late infections typically give rise to a more severe pathology and concomitant increased activation of the immune system. The increased activation of the immune system may dispose for the establishment of an autoimmune condition. This hypothesis would explain the apparent conflict in data indicating that viral infections may confer protection and susceptibility. The MIDIA study offers a unique possibility to test this hypothesis.

Viral infections have long been considered as triggers of T1D, and there are several lines of evidence implying virus infections in utero or early life in the aetiology of T1D . The high frequency of T1D in children with congenital rubella syndrome was the first indication of a viral involvement, and hinted towards the importance of the intra-uterine environment (Menser et al., 1978). Intra-uterine rubella infection is now rare in Scandinavia due to vaccination, but the incidence of T1D is high and continues to rise. Mumps and measles were also suspected of playing a role in T1D (Vuorinen et al., 1992), and a plateau in T1D incidence in Finland was also noted after measles, mumps and rubella vaccine was introduced (Hyöty et al., 1993). Measles vaccination was also suggested to be protective in a Swedish study (Dahlquist et al., 1991). Another interesting observation is that acute viral infections can be associated with disease onset (Elfaitouri et al., 2007; Frisk et al., 1992; Osame et al., 2007). There is also a seasonal correlation between periods of viral infections

**1.3.3 Bacterial colonization and virus infection in the intestines** 

**1.4 Viral infections as triggers of type 1 diabetes** 

Horman et al., 2004).

**1.3.2 Intestinal parasites** 
