Determination of autoantibodies in dogs with diabetes mellitus

Background and Aim: The classification of diabetes mellitus (DM) in dogs has been controversial as currently canine insulin-dependent DM is classified together with absolute insulin deficiency, non-insulin-dependent DM, and relative insulin deficiency. Studies on human autoantibodies evaluated in canines with DM, such as anti-glutamic acid decarboxylase (GAD65), anti-islet antigen 2 (IA2), and anti-zinc transporter isoform 8 (ZnT8), have been inconclusive. Thus, this study was designed to establish the serological profile of anti-GAD65, anti-IA2, and anti-ZnT8 antibodies in a group of dogs with and without DM. Materials and Methods: Sixty-one dogs, including 31 patients with DM (with and without insulin treatment) and 30 patients without DM (normal weight and obese), were included for determining autoantibodies using a human enzyme-linked immunosorbent assay (ELISA) detection system for type 1 DM. Results: This study found the presence of anti-IA2 antibodies in 58% of the sample (18/31 patients with DM); however, the presence of anti-GAD65 was not detected, and anti-ZnT8 was found in 3 (9.6%) patients with DM. Conclusion: This study showed a higher positive frequency of anti-IA2 antibodies in a sample of canine with DM, indicating that alterations in the signaling vesicle tyrosine phosphatase 2 lead to lower insulin release and thus to an increase in patients' glycemia. These preliminary results should be taken with caution and corroborated by a canine-specific assay when an ELISA is available for such determination.

Islet autoantibody level distributions in type 1 diabetes and their association with genetic and clinical characteristics

Positivity for islet autoantibodies is used for diagnosis of type 1 diabetes. However, the importance of the autoantibody level at diagnosis of type 1 diabetes is not clear. Here, we assessed the association of glutamate decarboxylase (GADA), islet antigen-2 (IA-2A) and zinc transporter 8 (ZnT8A) autoantibody levels, measured using radiobinding assays, on genetic and clinical characteristics at diagnosis of 1536 participants with diabetes who were positive for these autoantibodies. We show that GADA and IA-2A levels had bimodal distributions, but ZnT8A level did not. The comparison of genetic and clinical characteristics between high and low level categories showed high GADA level was associated with older age at diagnosis, female sex and HLA-DR3-DQ2, whereas high IA-2A level was associated with younger age of diagnosis, ZnT8A positivity and HLA-DR4-DQ8. We replicated our findings in an independent cohort of 427 people with type 1 diabetes where autoantibodies were measured using enzyme-linked immunosorbent assays. In conclusion, Islet autoantibody levels provide additional information over positivity in type 1 diabetes at diagnosis. The bimodality of islet autoantibody levels highlights the novel aspect of heterogeneity of type 1 diabetes which may have implications on prediction, treatment and prognosis.

Neutralizing Ljungan virus antibodies in children with newly diagnosed type 1 diabetes

Ljungan virus (LV), a Parechovirus of the Picornavirus family, first isolated from a bank vole at the Ljungan river in Sweden, has been implicated in the risk for autoimmune type 1 diabetes. An assay for neutralizing Ljungan virus antibodies (NLVA) was developed using the original 87–012 LV isolate. The goal was to determine NLVA titres in incident 0–18 years old newly diagnosed type 1 diabetes patients (n=67) and school children controls (n=292) from Jämtland county in Sweden. NLVA were found in 41 of 67 (61 %) patients compared to 127 of 292 (44 %) controls (P=0.009). In the type 1 diabetes patients, NLVA titres were associated with autoantibodies to glutamic acid decarboxylase (GADA) (P=0.023), but not to autoantibodies against insulin (IAA) or islet antigen-2 (IA-2A). The NLVA assay should prove useful for further investigations to determine levels of LV antibodies in patients and future studies to determine a possible role of LV in autoimmune type 1 diabetes.

Non-Genetically Encoded Epitopes Are Relevant Targets in Autoimmune Diabetes

Islet antigen reactive T cells play a key role in promoting beta cell destruction in type 1 diabetes (T1D). Self-reactive T cells are typically deleted through negative selection in the thymus or deviated to a regulatory phenotype. Nevertheless, those processes are imperfect such that even healthy individuals have a reservoir of potentially autoreactive T cells. What remains less clear is how tolerance is lost to insulin and other beta cell specific antigens. Islet autoantibodies, the best predictor of disease risk, are known to recognize classical antigens such as proinsulin, GAD65, IA-2, and ZnT8. These antibodies are thought to be supported by the expansion of autoreactive CD4+ T cells that recognize these same antigenic targets. However, recent studies have identified new classes of non-genetically encoded epitopes that may reflect crucial gaps in central and peripheral tolerance. Notably, some of these specificities, including epitopes from enzymatically post-translationally modified antigens and hybrid insulin peptides, are present at relatively high frequencies in the peripheral blood of patients with T1D. We conclude that CD4+ T cells that recognize non-genetically encoded epitopes are likely to make an important contribution to the progression of islet autoimmunity in T1D. We further propose that these classes of neo-epitopes should be considered as possible targets for strategies to induce antigen specific tolerance.

Regenerative neurogenic response from glia requires insulin driven neuron-glia communication

Understanding how injury to the Central Nervous System (CNS) induces de novo neurogenesis in animals would help promote regeneration in humans. Regenerative neurogenesis could originate from glia and glial Neuron-Glia antigen-2 (NG2) may sense injury-induced neuronal signals, but these are unknown. Here, we used Drosophila to search for genes functionally related the NG2 homologue kon-tiki (kon), and identified Islet Antigen-2 (Ia-2), required in neurons for insulin secretion. Alterations in Ia-2 function induced neural stem cell gene expression, injury increased ia-2 expression and induced ectopic neural stem cells. Using genetic analysis and lineage tracing, we demonstrate that Ia-2 and Kon regulate Drosophila insulin-like peptide 6 (Dilp-6), to induce glial proliferation and neural stem cells from glia. Ectopic neural stem cells can divide, and limited de novo neurogenesis could be traced back to glial cells. Altogether, Ia-2 and Dilp-6 drive a neuron-glia relay that restores glia, and reprograms glia into neural stem cells for regeneration.

Impaired Activated/Memory Regulatory T Cell Clonal Expansion Instigates Diabetes in NOD Mice

Regulatory T cell (Treg) insufficiency licenses the destruction of insulin-producing pancreatic b cells by auto-reactive effector T cells (Teffs), causing spontaneous autoimmune diabetes in non‑obese diabetic (NOD) mice. We investigated the contribution to diabetes of the TCR repertoires of naive regulatory T cells (nTregs), activated/memory Tregs (amTregs), and CD4⁺ Teffs from prediabetic NOD mice and normal C57BL/6 (B6) mice. NOD mice amTreg and Teff repertoire diversity was unexpectedly higher than that of B6 mice. This was due to the presence of highly expanded clonotypes in B6 amTregs and Teffs that were largely lost in their NOD counterparts. IL-2 administration to NOD mice restored such amTreg clonotype expansions and prevented diabetes development. In contrast, IL-2 administration only led to few or no clonotype expansions in nTregs and Teffs, respectively. Noteworthily, IL-2 expanded amTreg and nTreg clonotypes were markedly enriched in islet-antigen specific TCRs. Altogether, our results highlight the link between a reduced clonotype expansion within the activated Treg repertoire and the development of an autoimmune disease. They also indicate that the repertoire of amTregs is amenable to rejuvenation by IL-2.

Impaired Activated/Memory Regulatory T Cell Clonal Expansion Instigates Diabetes in NOD Mice

Regulatory T cell (Treg) insufficiency licenses the destruction of insulin-producing pancreatic b cells by auto-reactive effector T cells (Teffs), causing spontaneous autoimmune diabetes in non‑obese diabetic (NOD) mice. We investigated the contribution to diabetes of the TCR repertoires of naive regulatory T cells (nTregs), activated/memory Tregs (amTregs), and CD4⁺ Teffs from prediabetic NOD mice and normal C57BL/6 (B6) mice. NOD mice amTreg and Teff repertoire diversity was unexpectedly higher than that of B6 mice. This was due to the presence of highly expanded clonotypes in B6 amTregs and Teffs that were largely lost in their NOD counterparts. IL-2 administration to NOD mice restored such amTreg clonotype expansions and prevented diabetes development. In contrast, IL-2 administration only led to few or no clonotype expansions in nTregs and Teffs, respectively. Noteworthily, IL-2 expanded amTreg and nTreg clonotypes were markedly enriched in islet-antigen specific TCRs. Altogether, our results highlight the link between a reduced clonotype expansion within the activated Treg repertoire and the development of an autoimmune disease. They also indicate that the repertoire of amTregs is amenable to rejuvenation by IL-2.

Impaired Activated/Memory Regulatory T Cell Clonal Expansion Instigates Diabetes in NOD Mice

Regulatory T cell (Treg) insufficiency licenses the destruction of insulin-producing pancreatic b cells by auto-reactive effector T cells (Teffs), causing spontaneous autoimmune diabetes in non‑obese diabetic (NOD) mice. We investigated the contribution to diabetes of the TCR repertoires of naive regulatory T cells (nTregs), activated/memory Tregs (amTregs), and CD4⁺ Teffs from prediabetic NOD mice and normal C57BL/6 (B6) mice. NOD mice amTreg and Teff repertoire diversity was unexpectedly higher than that of B6 mice. This was due to the presence of highly expanded clonotypes in B6 amTregs and Teffs that were largely lost in their NOD counterparts. IL-2 administration to NOD mice restored such amTreg clonotype expansions and prevented diabetes development. In contrast, IL-2 administration only led to few or no clonotype expansions in nTregs and Teffs, respectively. Noteworthily, IL-2 expanded amTreg and nTreg clonotypes were markedly enriched in islet-antigen specific TCRs. Altogether, our results highlight the link between a reduced clonotype expansion within the activated Treg repertoire and the development of an autoimmune disease. They also indicate that the repertoire of amTregs is amenable to rejuvenation by IL-2.