Welcome to the Autoimmunity 2021 Congress Calendar

Background and Aims

The measurement of anti Glutamic acid decarboxylase autoantibodies (anti-GAD₆₅ AAb) is a hallmark in the serological diagnosis of type 1 diabetes (T1D). Until nowadays many of these tests have to be performed manually. A special assay architecture has to be chosen to yield highest possible assay characteristics. Here we report a fully automated assay for the measurement of anti-GAD₆₅ AAb.

Methods

The study involved 205 serum samples (clinically-defined type1 diabetes-patient samples n=100; Clinically-defined type2 diabetes-patient samples n=55; blood donor samples n=50. All sera were measured with a well established commercial ELISA (RSR ltd.) and by EliA anti-GAD65 (research use only, Thermo Fisher Scientific, Freiburg, Germany). Data analysis was performed using Analyse-IT for MS Excel.

Results

High agreement of the results was observed between the predicate ELISA and the novel EliA GAD65. Positive and negative agreement were found at 99% (95% CI=0.931-0.999). When comparing the quantitative results, a high correlation of results could be observed (r= 0949; 95% CI=0.934-0.961). Comparative clinical ROC analysis showed an AUC of 0.941 (95% CI=0.910-0.972) for the EliA GAD 65, and an AUC of 0.932 (95% CI=0.896-0.969). We thus conclude that the novel fully automated assay is comparable to its predicate device.

Conclusions

We found a high agreement of the novel Assay and the predicate device. The novel EliA GAD 65 showed high clinical sensitivity, comparable to the state of the art ELISA assay. We successfully showed a fully automated alternative to labour intensive manual ELISA for the serological identification of autoimmune diabetes type 1.

Background and Aims

Autoimmune Diabetes Mellitus (ADM) is an autoimmune metabolic disorder characterized by chronic hyperglycemia, presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, GAD 65, is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cells destruction and decline of pancreatic functions.

The human complement receptor type 1 (CR1) on B- and T-lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65-specific B cells from ADM patients by using chimeric molecules, containing an anti-CR1 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to bind selectively the anti-GAD65 specific B-cells by the co-crosslinking of the immunoglobulin receptor and CR1 and to deliver a suppressive signal.

Methods

Two synthetic peptide epitopes derived from GAD65 protein, and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro (Epitope prediction, Protein engineering, ELISA, FACS, ELISpot and Proliferation assay) and in vivo (NSG mice transfer) using PBMCs from diabetes patients.

Results

A reduction in the number of anti-GAD65 IgG antibody-secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of PBMCs from patients with ADM with engineered antibodies.

Conclusions

The constructed chimeric molecules are able to modulate selectively the activity of GAD65-specific B-lymphocytes and the production of anti-GAD65 IgG auto-antibodies by co-crosslinking of the inhibitory CR1 and the BCR.

Background and Aims

Several studies showed the difference in incidence of T1DM depending on the place of living. Much more children suffering from immunopathological diseases were urban residents. Based on above mentioned background we compared the regional distribution of the T1DM incidence in correlation to several urbanization-related factors.

Objective: identify urbanization-related factors affecting the incidence and prevalence of T1DM in children 0-14 years in RF.

Methods

The subject of the analysis was the incidence of T1DM “Children 0-14 years” group per 100 000 populations the period from 2008 to 2019.

Since among the classic urbanization-related factors are the indicators of air/water pollution, geographical density of public roads, number of public buses. Descriptive statistics included the Mean; Median [Q1; Q3]; and indications of the min-max values.We searched for the optimal models using the lowest value of the Akaike’s criterion.

Results

A significant multiplicative effect of the roads density, number of buses and air pollution on the incidence of T1DM was found. The number of buses and the paved roads geographic density have a multiplicative statistically reliable effect, and at the same time there is an independent effect of air pollution on the the incidence of T1DM (p>0.05).

Conclusions

The identified urbanization-related factors reliably contribute to the development and distribution of type I diabetes.

The main urban environmental factor contributing to the T1DM incidence confirmed during mathematical modeling is air pollution with solid dust particles, namely air emissions from the stationary sources, and impact of geographic density of hard-paved roads and the number of public buses.

Background and Aims

Type 1 Diabetes (T1D) caused by an autoimmune targeted destruction of the insulin-producing beta cells. The concordance for T1D in monozygotic twins and in the inbred NOD mice is far from 100%, despite genetic identity and shared environment during incidence peak years. This suggests stochastic determinants such as the never explored, post-zygotic mutations in the expanding antigen-specific T cell lineages, by analogy to their role in the expanding tumor lineage in cancer. We investigated somatic mutations as part of the cause of diabetes.

Methods

We used comparative genomic hybridization to identifiy somatic copy-number aberrations (CNAs) in DNA of memory CD4⁺ T cells from pancreatic lymph-nodes of diabetic NOD mice. To confirm the CNAs, we used the gold standard approach for CNA quantification, Multiplex ligation-dependent probe amplification. As controls, we examined DNA from lymphocytes expanded during normal host defense in mice infected with Leishmania major parasite. Also, we sequenced the TCR to determine the coloniality of these CD4⁺ memory cells.

Results

We found lymphocyte-exclusive mosaic somatic CNAs with highly non-random independent involvement of the same genes across different mice, some with an autoimmunity association. Lymphocytes involved in host defense were fewer and significantly smaller compared to those in autoreactive cells. Low T cell clonality for our samples suggests a pre-thymic formation of these CNAs.

Conclusions

Here, we explored a novel, unexplored phenomenon of a potential causal contribution of PZMs in autoreactive T cells in T1D pathogenesis. Our findings challenge the classical notions of autoimmunity and open conceptual avenues toward individualized prevention and therapeutics.

Background and Aims

The association between gut microbiota alterations and beta cell autoimmunity in type 1 diabetes (T1D) has been suggested by both animal model experiments and clinical studies. However, the adaptive immune system modulation exerted by the gut microbiota in the context of T1D remains poorly understood. To explore this issue, the islet beta cell-autoreactive B lymphocyte transgenic 116C-NOD mouse model, which surprisingly displays a decreased T1D incidence compared with NOD mouse, was used. Our objective was to analyze the effect of the 116C-NOD gut microbiota natural transfer on the immune phenotype, the intestinal microbial composition, and the progress of T1D in recipient NOD mice.

Methods

To achieve this goal, NOD mice were kept in isolation (isoNOD) or cohousing (coNOD) conditions regarding their transgenic 116C-NOD siblings. Fecal microbiome composition was analyzed using 16S rRNA gene sequencing. Immunological studies included B and T cell cytokine secretion profiling and population characterization after culture. T1D development was monitored by measuring glycosuria and glycaemia.

Results

Microbiome analysis revealed that coNOD harboured a different microbial profile compared with isoNOD mice. As for their immune phenotype, lymphocytes from coNOD mice developed a Th1/Th17 response, intermediate between the predominant Th1 pattern of isoNOD and the prevalent Th17 profile of 116C-NOD model. Furthermore, coNOD mice showed a significantly lower disease incidence than their isoNOD counterparts.

Conclusions

Taken together, these results suggest that 116C-NOD gut microbiota, previously tuned by the own 116C-NOD lymphocyte repertoire, may modify T1D development of recipient NOD mice through a Th status shift.

Background and Aims

Type 1 diabetes is due to the autoimmune destruction of pancreatic islet beta-cells. This study was aimed to assess whether the G3c monoclonal antibody (mAb), which triggers the glucocorticoid-induced TNFR-related (GITR) costimulatory receptor, promotes the expansion of regulatory T cells (Tregs) in diabetic-prone NOD mice and prevents diabetes onset.

Methods

G3c mAb was delivered via G3C hybridoma cells enveloped in alginate-based microcapsules that were grafted intraperitoneally into mice under general anesthesia. For each mouse, 1.5x10⁶ G3C hybridoma cells in 1 mL of microcapsules (G3C/cps) or 1 mL of empty microcapsules (e/cps) as a control, were grafted.

Results

Three weeks after G3C/cps graft, we observed expansion of conventional CD4⁺CD25⁺GITR^highFoxp3⁺ Tregs and non-conventional CD4⁺CD25^-/lowFoxp3^lowGITR^int/high (GITRsp) Tregs in the spleen. Expansion of both Treg subsets, including antigen-specific Tregs, was observed also in the pancreas of G3C/cps-treated as compared to e/cps-treated NOD mice. Moreover, the number of intact islets was higher in G3C/cps-treated than in e/cps-treated and untreated animals.

Consequently, all but two G3C/cps-treated mice (95%) did not develop diabetes and all but one survived till the end of the 24-week study. In comparison, only 50% of untreated NOD mice survived at the end of the study. Interestingly, the level of FoxP3 mRNA in the pancreas of G3C/cps-treated mice was 4000 fold higher than in the pancreas of e/cps and untreated diabetic mice.

Conclusions

In conclusion, long-term GITR triggering induces Treg expansion, thereby delaying/preventing diabetes development in NOD mice. This therapeutic approach may have promising clinical potential for the treatment of inflammatory and autoimmune diseases.

Background and Aims

Diabetic neuropathy (DN) is the most common complication of diabetes mellitus (DM). Corneal confocal microscopy (CCM) is a new noninvasive method that can help to study small nerve fibers in patients with DN.

To evaluate corneal small nerve fibers in patients with type 1 DM and DN, and dynamics of the nerve fibers by CCM during of the DM treatment.

Methods

20 patients with type 1 DM and DN were included. Median age 29 years [18.0; 45.0], the median HbA1c 8.5% [7.0; 10]. Neuropathy was assessed by Neuropathy Total Symptom Score (NTSS-9), Neuropathy Disability Score (NDS), CCM, lower limb electroneuromyography. After adjustment of glycaemia to HbA1c<7%, all tests were repeated.

Results

After reaching HbA1c<7%, there was a clinically significant improvement in NTSS-9 scores by 66.3% (p <0.001). There were increase in the number of nerve fibers (+16%), their thickness (+25.6%), the number of nerve branches (+32.4%) and nerve branch density – by 23.6% (p<0.001 for all parameters). The nerve fiber tortuosity decreased by 14.5%. Also, there was an increase of nerve conduction velocity of n. tibialis by 8.3% and n. suralis by 4.05% (p<0.05). Amplitudes of stimulation responses of these nerves increased by 9.9% and 8.5%, respectively (p<0.05).

Conclusions

The results allow us to propose the use CCM for diagnosis of DN. Improved glycemic control has a positive effect on the structural characteristics of corneal nerves.