**5. Effects of autoAbs on the launch of autoimmune processes and pancreas deficiency: Dynamics of spectra and prognostic value in preclinical diagnosis of T1D**

Today, autoantibodies (autoAbs) are the main biomarkers of diabetes mellitus. The presence of small concentrations of autoAbs in peripheral blood does not always indicate initiation of an autoimmune process, because the organism possesses a vast number of autoregulatory mechanisms. However, their collapse and significant elevation of Ab titers indicate a nearly 100% risk for diabetes in the foreseeable future. Many autoAb classes are currently known including GADA (65 and 67), IA-2Ab, INSab, HSPab, ZnT8ab, IAA, etc.

Each of these autoAbs has a prognostic value of its own and is associated with a definite type of diabetes (T1D, latent diabetes, fulminant diabetes, etc.). The factors initiating the appearance of autoAbs are also different. Among the immense diversity of causative factors, diabetogenic genes (classes I, II and III MHC) responding by activation to virus-induced inflammation (molecular mimicry) are of paramount importance. The results of in-depth studies on associativity between MHC II alleles and auto-Abs unequivocally suggest that in the present state of the problem generation of certain classes of auto-Abs and further progression of T1D can be predicted on the basis of genetic data even at the earliest stages of the disease.

### **5.1 GADA**

Glutamate decarboxylase catalyzes the conversion of glutamic acid into γ-aminobutyric acid and CO2 and plays a prominent role in the functional activity of the central nervous system (CNS). Its substrate (glutamic acid) is responsible for excitation, while its major metabolic product (γ-aminobutyric acid) is a key mediator of inhibition in brain neurons. The physiological role of this enzyme is confined to various aspects of insulin-dependent diabetes.

Two isoforms of GAD (GAD65 and GAD67) are encoded by two non-allelic genes localized on different chromosomes, more specifically, on the 2nd (GAD65) and 10th (GAD67) chromosomes. Both isoforms are actively expressed in CNS neurons. In human islet cells, GAD65 is a predominant isoform, while GAD67 is either present in negligibly small amounts or is not expressed at all. In contrast, in rat islet cells both GAD isoforms are expressed with a nearly equal efficiency, but the GAD67 isoform is predominant.

The prognostic significance of GADA is rather high. These autoAbs appear in circulating blood before other auto-Abs ( 10 – 15 years before the appearance of first clinical manifestations of T1D) and are detected in 70 – 90% of cases.

Risks for T1D and Ab titers


Likelihood of developing diabetes depending on the combination of epitope-specific GADA antibodies


For GAD antibodies, MID refers to epitopes within GAD65 amino acids 235–442, COOH refers to epitopes within GAD65 amino acids 436–585, NH2 refers to epitopes within GAD65 amino acids 1–100, while GAD67 refers to epitopes present in GAD6 (combinations with no relatives are not shown). (Buzzetti et al., 2007; Mayr et al., 2007)

#### **5.2 IA-2**

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

still unclear. Lumican, the key mediator in fibrosis-related processes, manifests an even higher degree of upregulation than Transcortin and is widely distributed in all body tissues. Its significant elevation may represent an acute response of renal tissues to high plasma levels of glucose and is also characteristic of nephropathies. However, being a convenient tool for predicting diabetic nephropathies, Lumican cannot predict associated diseases. It may be concluded from the above-said that Clusterin is the only candidate for a selective protein biomarker for T1D, because it emerges at early stages of the disease and is more

**5. Effects of autoAbs on the launch of autoimmune processes and pancreas** 

Today, autoantibodies (autoAbs) are the main biomarkers of diabetes mellitus. The presence of small concentrations of autoAbs in peripheral blood does not always indicate initiation of an autoimmune process, because the organism possesses a vast number of autoregulatory mechanisms. However, their collapse and significant elevation of Ab titers indicate a nearly 100% risk for diabetes in the foreseeable future. Many autoAb classes are currently known

Each of these autoAbs has a prognostic value of its own and is associated with a definite type of diabetes (T1D, latent diabetes, fulminant diabetes, etc.). The factors initiating the appearance of autoAbs are also different. Among the immense diversity of causative factors, diabetogenic genes (classes I, II and III MHC) responding by activation to virus-induced inflammation (molecular mimicry) are of paramount importance. The results of in-depth studies on associativity between MHC II alleles and auto-Abs unequivocally suggest that in the present state of the problem generation of certain classes of auto-Abs and further progression of T1D can be predicted on the basis of genetic data even at the earliest stages of

Glutamate decarboxylase catalyzes the conversion of glutamic acid into γ-aminobutyric acid and CO2 and plays a prominent role in the functional activity of the central nervous system (CNS). Its substrate (glutamic acid) is responsible for excitation, while its major metabolic product (γ-aminobutyric acid) is a key mediator of inhibition in brain neurons. The physiological role of this enzyme is confined to various aspects of insulin-dependent

Two isoforms of GAD (GAD65 and GAD67) are encoded by two non-allelic genes localized on different chromosomes, more specifically, on the 2nd (GAD65) and 10th (GAD67) chromosomes. Both isoforms are actively expressed in CNS neurons. In human islet cells, GAD65 is a predominant isoform, while GAD67 is either present in negligibly small amounts or is not expressed at all. In contrast, in rat islet cells both GAD isoforms are

The prognostic significance of GADA is rather high. These autoAbs appear in circulating blood before other auto-Abs ( 10 – 15 years before the appearance of first clinical

expressed with a nearly equal efficiency, but the GAD67 isoform is predominant.

manifestations of T1D) and are detected in 70 – 90% of cases.

**deficiency: Dynamics of spectra and prognostic value in preclinical** 

including GADA (65 and 67), IA-2Ab, INSab, HSPab, ZnT8ab, IAA, etc.

related to cause than effect.

**diagnosis of T1D** 

the disease.

**5.1 GADA** 

diabetes.

Risks for T1D and Ab titers

IA-2 belongs to type 1 membrane-bound proteins containing extracellular NH2-terminal glycosylated, membrane-bound and СООН-terminal cytoplasmic regions. The immune epitope of IA-2 is localized exclusively in the cytoplasmic region of IA-2 where its PTP (protein-tyrosine-phosphate)-like domain is the main recognition site for auto-Abs. The fact that the dominant T cell epitope of IA-2, also localized in the PTP-like region, is structurally similar to the VP7 region (VP7 is a major immunogenic protein of rotaviruses) provides additional evidence for the crucial role of the molecular mimicry mechanism in the pathogenesis of T1D.

IA-2β (also known as fogrin, PTP-NP, ICAAR and IAR) is structurally similar to IA-2. Its intracellular and extracellular domains are structurally identical (by 74 and 26%, respectively) to IA-2. IA-2β is predominantly localized in secretory vesicles of beta and some other neuroendocrine cells. Anti-IA-2β Abs are present in nearly 50% of patients with newly diagnosed T1D; their emergence is usually recorded several years before the appearance of the first clinical manifestations of the disease.

AutoAbs against the insulin antigen are detected in 70–90% of individuals as early as 10 – 12 years before the clinical stage of T1D.

Preclinical and Predictive Algorithms in Monitoring

**5.4 IAA - AutoAbs to insulin** 

reach very high levels.

preparation, also play a role.

30–60% of patients.

factors, etc.

Patients with Autoimmune Diseases and Their Relatives-at-Risks 207

Insulin is a peptide hormone produced by beta cells of pancreatic Langerhans islets. The main physiological role of insulin consists in reducing glucose levels in the blood; its absolute deficiency is the main cause of T1D.Anti-insulin Abs are indispensable constituent elements of blood sera of healthy individuals where their concentrations vary from 1 to 5 μg/ml. In patients with T1D, serum concentrations of anti-insulin autoAb IgG class can

Insulin antibodies are associated with many autoimmune pathologies including Graves' disease (40%), Hashimoto's disease (autoimmune thyroiditis) (20%), Addison's disease

Insulin-binding Ab are always present in the blood sera of insulin-treated patients. In patients with T1D, the therapeutic effect of insulin diminishes gradually with emergence of anti-insulin antibodies, especially after prolonged insulin therapy or administration of high daily doses of the hormone. Other factors, e.g., dosage form or purity of the hormonal

Seroconversion of insulin ABS is usually recorded as early as eight years before the onset and clinical diagnosis of T1D. However, after this period high antibody titers begin to decrease gradually up to the moment of their complete elimination and are detected in only

(40%), chronic hepatitis (36%), systemic lupus erythematosus (29%), etc.

Auto-Ab titers 10-year risk (% ± SE) HR (95% CI) P

II quartile 30 ± 13 0.7 (0.2–2.4) 0.55 III quartile 38 ± 17 0.8 (0.2–2.7) 0.68 IV quartile 77 ± 12 3.0 (1.1–8.1) 0.03

**6. Metabolome as applicable to T1D management at subclinical stages to** 

In a recent study, a comparison of blood sera from children with type 1 diabetes (T1D), non diabetic children and children without autoimmune antibodies revealed metabolic disturbances (significant reduction of serum levels of succinate, phosphatidylcholine, phospholipin and ketoleucine, drastic elevation of glutamate, etc.) in the T1D group. , (Bougnères et al., 2008) The true reason for these disturbances is difficult to establish, since all these changes can be associated, with an equal degree of probability, with asymptomatic damage of liver and body musculature, T1D, metabolic imbalance caused by environmental

High lysophosphatidylcholine levels are detected in patients' blood as early as several years before the first clinical manifestations of T1D. It should be noted, however, that the aforecited studies were performed on children, but not on adults with T1D; therefore, their

Notable elevation of blood sera levels of glutamate potentiates the activity of GAD65, the major autoimmune antigen for autoAbs. The figure below shows the dynamics of the "glutamine–GABA–GADA" sequence. As can be seen, neither GADA and IAA, nor unlimited

I quartile 45 ± 16 1\*

(Catherine Pihoker et al., 2005; Heli et al., 2009)

**prevent or minimize the imbalance** 

clinical significance is ambiguous.


Likelihood of developing diabetes depending on combination of epitope-specific IA-2 antibodies


For IA-2 antibodies, IA-2β refers to epitopes found in the PTP region of IA-2β and IA-2, PTP refers to epitopes found in the PTP region of IA-2 (but not IA-2β), while JM refers to epitopes within the IA-2 juxtamembrane region in amino acids 601–682 (combinations with no relatives are not shown). (Kordonouri et al., 2010; Kawasaki E et al., 2003; Hanifi-Moghaddam et al., 2003)

#### **5.3 ICA**

The target antigen for pancreatic islet cell antibodies has not yet been finally identified. In all probability, it represents a heterogeneous cluster of antigens expressed in beta cells. In contrast to IA-2ab, ICA is a polyclonal antibody able to interact with all populations of islet cells (α, β, γ, δ, PP) and other auto-antigens (sialoglucoconjugate, GAD, IA-2A, etc.).

Antibodies to islet cells are found in 85-90% of patients with onset T1D (cf. 0.5% in unaffected individuals) during the very first week after clinical diagnosis. Four weeks thereafter, their incidence does not exceed 50%. In patients with one-year history of T1D, antibodies to beta cells are present in only 10-20% of cases.

Similar to other cell-related Abs, ICA does not play any crucial role in beta cell degradation, but is one of key markers of cell-mediated autoimmunity. Its detection in blood serum suggests latent autoimmune diabetes (LADA) and slow degradation of beta cells.The prognostic capacity of ICA is neither high, nor low and is usually manifested 12 years prior to T1D development. Its incidence at the clinical diagnosis stage varies from 60 to 80%. Correlation between antibody titers and risks for T1D over a period of 7 years. (Achenbach et al., 2004; Williams et al., 2002)

#### **5.4 IAA - AutoAbs to insulin**

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

Auto-Ab titer 10-year risk (% ± SE) HR (95% CI) P

II quartile 74 ± 15 6.0 (1.6–22.4) 0.008 III quartile 84 ± 10 5.9 (1.7–21.1) 0.006 IV quartile 71 ± 15 4.8 (1.3–17.9) 0.02

Likelihood of developing diabetes depending on combination of epitope-specific IA-2

IA-2ab

n (T1D cases) 10-year risk

+ + + 13 (8) 81% + + − 12 (10) 100%

− + + 8 (3) 34% − + − 10 (4) 44% − − + 9 (2) 41%

For IA-2 antibodies, IA-2β refers to epitopes found in the PTP region of IA-2β and IA-2, PTP

The target antigen for pancreatic islet cell antibodies has not yet been finally identified. In all probability, it represents a heterogeneous cluster of antigens expressed in beta cells. In contrast to IA-2ab, ICA is a polyclonal antibody able to interact with all populations of islet

Antibodies to islet cells are found in 85-90% of patients with onset T1D (cf. 0.5% in unaffected individuals) during the very first week after clinical diagnosis. Four weeks thereafter, their incidence does not exceed 50%. In patients with one-year history of T1D,

Similar to other cell-related Abs, ICA does not play any crucial role in beta cell degradation, but is one of key markers of cell-mediated autoimmunity. Its detection in blood serum suggests latent autoimmune diabetes (LADA) and slow degradation of beta cells.The prognostic capacity of ICA is neither high, nor low and is usually manifested 12 years prior to T1D development. Its incidence at the clinical diagnosis stage varies from 60 to 80%. Correlation between antibody titers and risks for T1D over a period of 7 years. (Achenbach

refers to epitopes found in the PTP region of IA-2 (but not IA-2β), while JM refers to epitopes within the IA-2 juxtamembrane region in amino acids 601–682 (combinations with

no relatives are not shown). (Kordonouri et al., 2010; Kawasaki E et al., 2003; Hanifi-

cells (α, β, γ, δ, PP) and other auto-antigens (sialoglucoconjugate, GAD, IA-2A, etc.).

I quartile 20 ± 14 1\*

Epitope combination

IA-2β PTP JM

+ − + 1 (1) + − − 4 (3)

− − − 4 (1)

antibodies to beta cells are present in only 10-20% of cases.

antibodies

Moghaddam et al., 2003)

et al., 2004; Williams et al., 2002)

**5.3 ICA** 

Insulin is a peptide hormone produced by beta cells of pancreatic Langerhans islets. The main physiological role of insulin consists in reducing glucose levels in the blood; its absolute deficiency is the main cause of T1D.Anti-insulin Abs are indispensable constituent elements of blood sera of healthy individuals where their concentrations vary from 1 to 5 μg/ml. In patients with T1D, serum concentrations of anti-insulin autoAb IgG class can reach very high levels.

Insulin antibodies are associated with many autoimmune pathologies including Graves' disease (40%), Hashimoto's disease (autoimmune thyroiditis) (20%), Addison's disease (40%), chronic hepatitis (36%), systemic lupus erythematosus (29%), etc.

Insulin-binding Ab are always present in the blood sera of insulin-treated patients. In patients with T1D, the therapeutic effect of insulin diminishes gradually with emergence of anti-insulin antibodies, especially after prolonged insulin therapy or administration of high daily doses of the hormone. Other factors, e.g., dosage form or purity of the hormonal preparation, also play a role.

Seroconversion of insulin ABS is usually recorded as early as eight years before the onset and clinical diagnosis of T1D. However, after this period high antibody titers begin to decrease gradually up to the moment of their complete elimination and are detected in only 30–60% of patients.


(Catherine Pihoker et al., 2005; Heli et al., 2009)
