Welcome to the CORA 2023
Interactive Program

Background and Aims

Objective An increased permeability of Intestinal barrier (IB), known as “leaky gut”, has been demonstrated in patients with intestinal disorders and in patients with autoimmune diseases involving organs far from the intestine, as in patients with Hashimoto’s thyroiditis (HT). The indirect measure of IB permeability in patients with HT, even in a polyautoimmunity context, represents the aim of our study.

Methods

Methods The study group encompassed 93 patients bearing HT (median age=48 years); 33 of them associated another non-endocrine autoimmune disorder (HT+POLY) [13 gastric atrophy (HT+GA), 13 vitiligo (HT+V) and 7 celiac disease (HT+CD)]. The evaluation of gut permeability was performed by dosing serum zonulin and LPS. The serum of patients was stored at -20°until the samples were analyzed by ELISA kits.

Results

Result Zonulin and LPS were higher in patients HT+POLY than in patients with isolated HT (p<0.0001 and 0.0004, respectively). The highest concentrations of zonulin as compared to HT may be observed in HT+CD (p<0.0001), followed by HT+GA (p<0.01). On the contrary, the highest concentration of LPS as compared to HT may be observed in HT+V (p<0.01), followed by HT+GA (p<0.05). In the whole sample and in patients with isolated HT, zonulin and LPS concentrations significantly correlated (p<0.0001;r=0.4431 and r=0.4409, respectively), a correlation lost in patients with HT+POLY.

Conclusions

Conclusions Increased levels of zonulin and LPS in patients with polyautoimmunity, as compared with HT patients, suggest an increased permeability and a more severe systemic inflammatory state even when the additional disease does not involve directly the gastrointestinal tract.

Background and Aims

Immunosuppressive CD4+ T regulatory (Treg) cells prevent autoimmunity and functional T cell-dendritic cell (DC) interactions contribute to reciprocal stimulation leading to DC maturation that results in T cell production of interleukin-2 (IL-2) required to sustain Tregs. However, autoreactive T cells are activated by agonistic cytokines such as interferon-gamma (IFN-γ) and interleukin-12p40 (IL-12p40) that positively regulate one another. Importantly, IL-12p40 inhibits IL-12p70 activity including Treg cell function and activation of IL-12p40 is regulated by calcium-dependent signalling in DCs that involves the Src family kinase member, c-Src.

We have previously reported that a 10 mer peptide, RSKAKNPLYR, inhibits c-Src activity and the aim of the present study was to examine its effect on T cell-DC interactions when conjugated to a branched lipid unit comprising dodecanoic acid residues to facilitate stability and cell entry of the 10 mer component.

Methods

The conjugated peptide, designated IK14004, was made using solid phase peptide synthesis with Fmoc protected building blocks and product structures were confirmed by mass spectroscopy and amino acid analysis. Cell-based studies (enzyme-linked immunoassays and flow cytometry) were performed on immune cells isolated from healthy volunteers and kinase profiling was performed using non-cell-based assays.

Results

IK14004 enhances production of IL-2/IL-12p70 by T cells and expands the Treg cell population while destabilising DCs and inhibiting production of IL-12p40 and IFN-γ consistent with inhibition of c-Src activity and the calcium-dependent signalling pathway.

Conclusions

This small molecule inhibitor offers an opportunity to gain further insight into the complexity of T cell-DC interactions relevant to autoimmunity.

Background and Aims

To examine the frequencies of peripheral blood(PB) B-cell subsets across different PsA disease phases, evaluating their correlation with synovial tissue (ST) inflammation.

Methods

150 patients fulfilling the CASPAR criteria(81 naive to therapy,45 c- and/or b-DMARDs resistant and 24 in clinical and US sustained remission, respectively) underwent US-guided ST biopsy and PB withdrawal.22 patients with psoriasis (PsO) and arthralgia were included as comparison group.All ST FFPE specimens were stained with H&E and classified by a pathologist using a H&E based semiquantitative score (KSS).Frequencies of PB B-cell subpopulations were determined by FACS using the CD27/IgD classification as follows: naïve B-cells,IgM memory,switched memory, late memory,plasmablasts and plasmacells.

Results

KSS was significantly different across disease phases.Specifically,KSS was contingent on disease phase being lower in remission compared to naive (p=0.02) and resistant PsA (p<0.001),while there was no difference between remission PsA and at risk PsO group.ST of c- and/or b-DMARDs resistant PsA was enriched of plasmacells than remission (p=0.03).Considering B-cell subpopulations,PB of c- and/or b-DMARDs resistant PsA was enriched of plasmablasts and plasmacells compared to PsO at risk (p=0.01 and p=0.001,respectively).Conversely,c- and/or b-DMARDs resistant PsA showed,at PB level, lower rates of switched memory and naïve B-cells compared to PsO at risk (p= 0.02 and p=0.039,respectively).Finally, stratifying PsA based on KSS category, remission PsA with persistent high grade synovitis(KSS ≥ 5) showed lower rates of PB plasmablasts than remission PsA with low grade residual synovial inflammation(p=0.045).

Conclusions

Disease state across PsA course significantly impacts PB B-cell subsets distribution mirroring ST residual inflammation at the time of sustained disease remission.

Background and Aims

Calprotectin (S100A8/S100A9, MRP8/MRP14) in plasma has been shown to be more sensitive than Erythrocyte Sedimentation Rate (ESR) or C-Reactive Protein (CRP) in reflecting inflammatory activity in patients with rheumatoid arthritis (RA).

The aim of this study was to explore the robustness of the laboratory examination and the correlation with clinical examinations of inflammation.

Methods

Plasma samples from early (n=220, mean disease duration 7.2 months) and established RA (n=177, mean disease duration 10.0 years) patients were analyzed for calprotectin levels at baseline and after 1, 2, 3, 6 and 12 months by use of either Enzyme-linked immunosorbent assay (ELISA, Calpro AS) or fluoroenzyme immunoassay (FEIA, measured on EliA platform). Clinical measures as number of swollen joints and examiner’s global score (EGA) by use of visual analogue scale (VAS) (score 0-100) were included as well as Ultrasound performed by an experienced sonographer.

Results

The two Calprotectin methods showed high correlation (Spearman correlation 0.91 and 0.96 for early respective established RA). When comparing number of patients in remission having normal calprotectin levels compared to normal CRP levels, there was a higher percentage of agreement for Calprotectin compared to CRP, 93-96% vs 71-85% depending on timepoint and remission criteria.

Conclusions

Calprotectin has been shown to be a better marker of inflammation than the commonly used inflammatory markers and may therefore be widely included in clinical laboratories. The present study supports the robustness of the analyses, showing similar calprotectin measures across different analytical methods and better correlation with remission than CRP in RA patients.

Background and Aims

Studies have linked HHV-6 with thyroid autoimmunity, yet the exact mechanisms of HHV-6 involvement remain unclear. We propose that two poorly studied HHV-6 proteins – U12 and U51, may contribute to autoimmunity. They are homologous to chemokine receptors (G-protein coupled receptors), can be expressed on cell surfaces, and can influence viral replication. By conducting molecular and immunological investigations we aim to elucidate the potential role of U12/U51 in thyroid autoimmunity.

Methods

Thyroid tissue and plasma samples from 54 AIT patients (harboring HHV-6 in the thyroid) were investigated. RNA was isolated from thyroid tissues, used for cDNA synthesis, and nPCR to detect U12, U51 mRNA. FFPE thyroid tissues were microscoped to visualize U12/U51. U12 and U51 antibodies were detected with the Luminex system

Results

44% of thyroid tissues harbored U12/U51 mRNA. Thyroid tissues with mRNA harbored significantly higher viral loads (1998 vs 238 viral copies/10⁶ cells, p<0,0001). Nearly all AIT patients were shown to harbor U12/U51 antibodies, regardless of mRNA presence. Microscopy revealed the colocalization of HHV-6 and U12/U51 in the thyroid.

Conclusions

The combination of the presence of U12/U51 mRNA and viral protein-specific antibodies with the visualization of both HHV-6 and its GPCRs in the thyroid suggests that both could be involved in autoimmunity development/exacerbation. HHV-6 and U12/U51 could enhance thyroid cell destruction, autoantigen release, and inflammation through viral replication enhancement or through direct antibody binding and subsequent immune response against U12/U51

Acknowledgments. EU Horizon 2020 project “Reducing networking gaps between RSU and internationally leading counterparts in viral infection-induced autoimmunity research (VirA)” (No952376)

Background and Aims

Leukocyte transendothelial migration is one of the most important steps in launching an inflammatory immune response. However, overstimulated of disregulated mode of the leukocyte transmigration from blood to the tissus can lead to devastating autoimmune diseases. Based on previously reported trioxotetrahydropyrimidin integrin inhibitors and in silico calculations, we designed several molecules with a modified barbituric acid scaffold and tested them in vitro.

Methods

In silico calculations and modeling, organic synthesis, analytical chemistry (NMR and Mass spectroscopy), leukocyte transmigration flow assay, adhesion molecules activity assay kits, six mouse inflammatory/autoimmune diseases models (Nonalcoholic fat liver, IBD, arthritis, acute respiratory syndrome, brain form of lupus and multiple sclerosis), toxicity studies, structure-activity relationship study, blood biochemistry, histochemistry and target validation study.

Results

One of the molecules: GT-73, completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC₅₀=2.4 µM). In vivo, GT-73 exhibited significant therapeutic effects in all tested in vivo models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. GT-73 was active in pharmacological relevant doses (10-30 mg/kg) and in several routes of administration. Most important that GT-73 was active orally. The mechanism of GT-73 action was validated (reversible covalent inhibition of active dimer form of PECAM-1). The critical for the activity functional groups were also determined: tert-butyl and methyl ester.

Conclusions

GT-73 might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.