**2.3 Pathogenesis**

*Genetic susceptibility* plays an important pathogenetic role in type 1 diabetes mellitus, with the HLA -DR and –DQ genes explaining about 50% of the risk. The different penetrance of these genes can partially explain the role of environmental factors. Whereas differences in incidence between populations may be due to genetic susceptibility or protection genes, the increasing incidence is to be ascribed to environmental factors. Studies aimed to analyze the temporal changes in the frequency of genotypes associated with type 1 diabetes susceptibility reported a decreasing frequency of those higher-risk HLA genotypes (DRB1 03-DQA1\*0501-DQB1\*0201/DRB104-DQA1\*0301-DQB1\*0302) between recently diagnosed patients as compared to those diagnosed 50 years before (Hermann et al., 2003). Therefore the increasing incidence of type 1 diabetes could be explained by a more permissive environment, exerting in increased penetrance of low/moderate risk genotypes or in interplay between environmental factors and other non-HLA genes (Nejentsev et al., 2007, Todd et al., 2007). On the other hand, several environmental factors such as dietary habits, sedentary lifestyle, climate changes, pollution, viral (also maternal) and other infectious disease frequency and type, have changed over the past years and deserve attention (Oikarinen et al., 2011).

The *immune-mediated -cell destruction* occurs over several years and exerts in progressive insulin deficiency, leading to various degrees of hyperglycemia up to severe metabolic derangement , i.e. diabetic ketoacidosis (Devendra et al., 2004).

Studies in both humans and animal models are trying to clarify the specific antigenic targets involved in the islet cell autoimmunity. Islet cell antibodies (ICA) detected by immunofluorescence, were firstly isolated in patients with diabetes mellitus and autoimmune polyglandular syndrome (Bottazzo et al., 1974). These autoantibodies are transient, being observed in 70-80% of newly-diagnosed cases and tend to disappear thereafter. Their positivity in subjects at risk of diabetes (i.e. first-degree relatives) represents a useful means to predict the future development of the disease.

Anti-insulin autoantibodies (IAA) can be detected both by radioimmunoassay (RIA) or by ELISA, but the first method is recommended. IAA positivity is inversely related to age at diabetes diagnosis (81% in patients younger than 10 and 61% in older ones), and is higher and adolescent males (Williams et al., 2003). IAA are the first autoantibodies to became positive, and they can later decline. Interestingly, substitutive insulin therapy can be followed by an immune response against itself, and this subtype of insulin antibodies can be distinguished from anti-insulin autoantibodies.

In 1990 Baekkeskov and co-workers reported that the 64,000 M (R) molecule previously defined as an antigenic target of Type 1 diabetes was the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD) (Baekkeskow et al., 1990). GAD is not expressed exclusively on -cells, but also in other islet cells. Anti-GAD autoantibodies (GADA) can be detected both by RIA or by ELISA. The prevalence of anti-GAD autoantibodies positivity is 84%, and is positively related to age and female sex. Their peak level can be reached after diabetes diagnosis and persist longer than anti-islet cell antibodies, making them a useful marker especially for adult patients.

In 1994 a cDNA coding a 548 aminoacid protein named ICA-512 was described as a major target of humoral immunity by screening an islet c-DNA expression library with patients' sera (Rabin et al., 1994). Moreover it has been reported that IA-2, a 979 aminoacid transmembrane protein of the tyrosine phosphatase family, is a major autoantigen in type 1 diabetes. IA-2 is a intrinsic membrane protein of secretory granules neuroendocrine cells, like pancreatic islets. IA-2 autoantibodies (IA-2A) can be detected by RIA as well as by ELISA. Recently a not radio-isotopic method (time-resolved immunofluorometric assay (TR-IFMA) showed comparable results with RIA. The prevalence of IA-2A has been reported about 73%, and no correlation with age was found (Tsirogianni et al., 2009).

Recently the cation efflux transporter 8 (ZnT8) has been identified as a novel target autoantigen in patients with type 1 diabetes. Autoantibodies to ZnT8 (ZnT8 A) are detectable in about 70% of newly diagnosed patients, independent of age (Achenbach et al., 2009). Patients presenting with a single islet cell autoantibody were also positive for ZnT8 A, suggesting that they could be a marker for type 1 diabetes risk stratification. Three variants of ZnT8 A have been recognized: 1) ZnT8RA (arginine 325 zinc transporter 8 autoantibody), 2) ZnT8WA (tryptophan 325 zinc transporter 8 autoantibody), 3) ZnT8QA (glutamine 325 zinc transporter 8 autoantibody). These 3 ZnT8 variants precede T1DM clinical onset and are all detectable by radio-binding assay (Andersson et al., 2011).

#### **2.4 Diagnosis and treatment**

Diagnosis of type 1 diabetes is based on symptoms of hyperglycemia: polyuria, polydipsia, with mild symptoms up to severe ketoacidosis. After intravenous fluid, insulin and salt replacement for metabolic imbalance recovery, treatment of type 1 diabetes consists of lifelong substitutive subcutaneous insulin therapy, together with correct dietary habits, selfmanagement of the disease and regular physical activity, as result of a prolonged educational intervention starting at the time of clinical diagnosis (Maffeis & Pinelli, 2008, Bangstad et al., 2007). Recognition, management and prevention of hypoglycemic episodes as well as hyperglycemic spikes is mandatory. Continuous education implementation starting at the time of clinical diagnosis, designed for children, adolescents and their parents, is necessary thereafter (Weinzimer et al., 2005).

#### **2.5 Follow-up**

4 Autoimmune Disorders – Current Concepts and Advances from Bedside to Mechanistic Insights

The incidence of Type 1 diabetes is increasing worldwide and may double the burden of the disease in youngest children by 2020. Large collaborative studies like DiaMond and EURODIAB Registries demonstrated that one century ago childhood diabetes was rare and fatal, while at the end of the century a steady increase in several parts of the world has been observed. In particular, DiaMond Project reported the trend in incidence of Type 1 diabetes from 1990 to 1999; over this period the average annual increase in incidence was 2.8%, with a slight higher rate in the last 5 year-period as compared to the first 5-year period. Similarly, EURODIAB Study reported a 3.9% annual increase from 1989-2003 (Vehil & Dabelea, 2011). The rising incidence of Type 1 diabetes over the past decades is too quick to be attributed to an increased genetic susceptibility, since the proportion of newly-diagnosed patients

*Genetic susceptibility* plays an important pathogenetic role in type 1 diabetes mellitus, with the HLA -DR and –DQ genes explaining about 50% of the risk. The different penetrance of these genes can partially explain the role of environmental factors. Whereas differences in incidence between populations may be due to genetic susceptibility or protection genes, the increasing incidence is to be ascribed to environmental factors. Studies aimed to analyze the temporal changes in the frequency of genotypes associated with type 1 diabetes susceptibility reported a decreasing frequency of those higher-risk HLA genotypes (DRB1 03-DQA1\*0501-DQB1\*0201/DRB104-DQA1\*0301-DQB1\*0302) between recently diagnosed patients as compared to those diagnosed 50 years before (Hermann et al., 2003). Therefore the increasing incidence of type 1 diabetes could be explained by a more permissive environment, exerting in increased penetrance of low/moderate risk genotypes or in interplay between environmental factors and other non-HLA genes (Nejentsev et al., 2007, Todd et al., 2007). On the other hand, several environmental factors such as dietary habits, sedentary lifestyle, climate changes, pollution, viral (also maternal) and other infectious disease frequency and type, have changed over the past years and deserve attention

insulin deficiency, leading to various degrees of hyperglycemia up to severe metabolic

Studies in both humans and animal models are trying to clarify the specific antigenic targets involved in the islet cell autoimmunity. Islet cell antibodies (ICA) detected by immunofluorescence, were firstly isolated in patients with diabetes mellitus and autoimmune polyglandular syndrome (Bottazzo et al., 1974). These autoantibodies are transient, being observed in 70-80% of newly-diagnosed cases and tend to disappear thereafter. Their positivity in subjects at risk of diabetes (i.e. first-degree relatives) represents

Anti-insulin autoantibodies (IAA) can be detected both by radioimmunoassay (RIA) or by ELISA, but the first method is recommended. IAA positivity is inversely related to age at diabetes diagnosis (81% in patients younger than 10 and 61% in older ones), and is higher and adolescent males (Williams et al., 2003). IAA are the first autoantibodies to became positive, and they can later decline. Interestingly, substitutive insulin therapy can be followed by an immune response against itself, and this subtype of insulin antibodies can be

*-cell destruction* occurs over several years and exerts in progressive

carrying the highest-risk HLA genotype (HLA DR3/DR4) seems unchanged.

**2.2 Epidemiology** 

**2.3 Pathogenesis** 

(Oikarinen et al., 2011). The *immune-mediated* 

distinguished from anti-insulin autoantibodies.

derangement , i.e. diabetic ketoacidosis (Devendra et al., 2004).

a useful means to predict the future development of the disease.

The most serious problem related to pediatric type 1 diabetes is the risk, even in young adulthood, of microvascular and macrovascular complications, i.e. retinopathy, nephropathy, neuropathy, cardiovascular and cerebrovascular diseases (Donaghue et al., 2007). The key role of good glycaemic control to prevent diabetes-related complications has been firmly established by the Diabetes Control and Complications Trial Study, which demonstrated the protective role of intensive insulin treatment (DCCT, 1993). Sustained

Autoimmune Disorders Associated to Type 1 Diabetes Mellitus in Children and Adolescents 7

*Genetic factors* play an important pathogenetic role, as demonstrated by a concordance rate of 85% in monozygotic twins and by familiar aggregation. HLA-DQ genes, in particular DQ2 variant (alleles DQA1\*05/DQB1\*02) and DQ8 variant (alleles DQA1\*03/DQB1\*0302) are strongly associated to CD. Beside the HLA genes (i.e. COELIAC 1, on chromosome p21), other non-HLA genes are recognized to confer additional susceptibility: COELIAC 2, on chromosome 5q31-33, which contains cytokine gene clusters; COELIAC 3, on chromosome 2q33, which codes the negative co-stimulatory molecule CTLA4; COELIAC 4, on chromosome 19p13.1, which contains the myosin IXB gene variant encoding a myosin that

As regards *pathophysiology* od CD, it has been demonstrated that gluten peptides, which are resistant to digestion by gastric and pancreatic enzymes, after crossing intestinal epithelium, are deaminated by tissue transglutaminase and then presented by DQ2+ or DQ8+ antigenpresenting cells to gluten-specific CD4+ T cells. These cells once activated drive a Th1 response, characterized by production of pro-inflammatory cytokines, and responsible for the development of celiac lesions, i.e. lamina propria infiltration of inflammatory cells, crypt

The clinical range of celiac disease has a wide spectrum, from asymptomatic to severe malnutrition, with gastrointestinal and extra-intestinal manifestations. The most common feature of celiac disease includes gastrointestinal symptoms (i.e. abdominal pain, increased frequency of bowel movements), weight loss, bone disease, various degree of anemia and

Different subtypes of celiac disease have been described. Symptomatic or classic celiac disease means typical gastrointestinal symptoms with severe malabsorption syndrome. The term atypical celiac disease is applied to cases with mild or absent gastrointestinal symptoms (colitis or irritable bowel), and characterized by extra-intestinal manifestations, including iron deficient anemia, osteoporosis, failure to thrive. In both cases villous atrophy in observed during endoscopy or intestinal biopsies (Alaedini & Green, 2005). More recently it has been suggested to define celiac disease as silent, minor or major. Silent celiac disease is referred to asymptomatic subjects, sometimes relatives of patients with known celiac disease, or subjects eventually found to be positive at screening procedures. Minor celiac disease is referred to subjects with transient symptoms (dyspepsia, irritable bowel syndrome without malabsorption), anemia, cryptic hypertransaminasemia, infertility, peripheral and central neurological disorders, osteoporosis, dental enamel defects, failure to thrive, dermatitis herpetiformis. Major celiac disease is referred to patients with major

The mechanism underlying the severity of clinical presentation at present remains unknown. Researchers have shown that neither the degree of duodenal villous atrophy nor the extent of visible enteropathy assessed by capsule endoscopy correlates with presentation

The recognition of a pre-celiac disease state is usually retrospective and this condition has

*Potential celiac disease* is characterized by positive antibodies but normal mucosa; there is no evidence to support managing these patients with a gluten-free diet. A higher prevalence of potential CD was found in patients with type 1 diabetes, and this observation may be

alters actin remodeling (Di Sabatino & Corazza, 2009).

**3.3 Clinical presentation and diagnosis** 

weakness.

hyperplasia and villous atrophy (Di Sabatino & Corazza, 2009).

gastrointestinal symptoms (Di Sabatino & Corazza, 2009).

(Di Sabatino & Corazza, 2009).

been termed latent celiac disease.

chronic hyperglycaemia and acute blood glucose fluctuations have a deleterious effect on the metabolic mechanisms involved in the development of microangiopathy, such as protein glycation and oxidative stress. In particular, glucose variability from peaks to nadir, with upward as in the postprandial periods, and nadirs, as in the interprandial periods activates the oxidative stress (Monnier & Colette, 2008). As regards pediatric diabetes, despite better insulin preparations and strict self-management of the disease few children and adolescents maintain mean glycated hemoglobin A1c (HbA1c) levels within the normal ranges, with serious impact on metabolic control and subjects' caregiver quality of life (Rewers et al, 2009).
