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Background and Aims

Radiographic axial spondyloarthritis/ankylosing spondylitis (AS) is a chronic inflammatory arthritis characterized by sacroiliitis, enthesitis and a marked propensity for sacroiliac joint and spinal fusion. Interleukin 35 (IL-35) is the newest identified member of the interleukin-12 cytokine family, a unique group of heterodimeric cytokines including IL-12, IL-23, IL-27, and IL-35, which share structural similarities but mediate diverse immunological functions.Accordingly, the objective of of the present study was to examine the impact of IL-35 in AS patients and assess the association between serum IL-35 levels, disease activity and radiographic progression including bone erosions.

Methods

Forty AS patients diagnosed according to the ASAS/EULAR criteria, and 20 controls were recruited. Serum IL-35 levels were measured by ELISA. Disease activity and health outcome measures assessed included: Bath Ankylosing Spondylitis Disease Activity Index (BASDAI),Bath Ankylosing Spondylitis Functional Index(BASFI), Bath Ankylosing Spondylitis Metrology Index(BASMI), Ankylosing Spondylitis Disease Activity Score-C reactive protein(ASDASCRP), Maastricht Ankylosing Spondylitis Enthesitis Score and Patient Global Assessment score. Radiographic assessment of the spine and sacroiliac joints was done using the Modified Stoke Ankylosing Spondylitis Spinal Score.

Results

The mean serum IL-35 level in AS patients was significantly less compared to controls, 14.68±2.72 pg/ml versus 30.62±6.04 pg/ml; p< 0.001. A significant correlation was found between serum IL-35 levels and BASDAI(r=-0.363, p=0.021), BASMI r=-0.605, p< 0.001), and ASDASCRP(r= -0.597, p<0.001) respectively. Higher IL-35 levels were associated with less bony erosions.

Conclusions

IL- 35 has a protective role in AS; it is associated with less disease activity and better health outcomes. IL-35 is thus a potential therapeutic target for AS.

Background and Aims

It is known that the presence of some non- criteria antiphospholipid anibodies (n-c aPL) is connected with thrombosis and pregnancy complications. Some n-c aPL are considered as significant for diagnosis of primary antiphospholipid syndrome (pAPS).

The aim of the study was to determine the prevalence of non-criteria antiphospholipid anibodies in a group of patients with pAPS

Methods

The study involved 26 (16 f,10 m) of APS patients (pts) observed for a longterm in a University Clinic. All pts fulfilled the criteria for classification of APS . The mean age of the pts was: 40,81± 13,42 (range 18-66), the duration of the disease was 9,2 ± 8,83 years (range 1-37).

The presence of Ab was detected in patients’ serum using the commercially available tests: aPL-immunodot assay Anti-Phospholipid 10 Dot, for the qualitative detection of IgG or IgM antibodies. Statistical data analysis was performed using Statistica v13.0

Results

N-c aPL were detected relatively often in p APS pts (a-phosphatidylserine IgG 65.4% of pts; a-prothrombin IgM 57,7%). A-prothrombin IgM were detected significantly more often than a a-CL IgM and a a-phosphatydylserin IgG were detected significantly more often than a a-B2GPI IgG.

The clinical symptoms observed in the pts are: thrombosis 69,2%; stroke 30,8%; pulmonary embolism 30,8%; pregnancy complications 26,7%, migraine 26,9%, livedo reticularis 69,2%, infarct 15,4% and seizures 7,7%. Migraine, seizures and pregnancy complications significantly correlated with some n-c aPL.

Conclusions

N-c aPL can be a valuable tool for accurate diagnosis of p APS and my help in making an appropriate decision concerning the treatment

Background and Aims

Pathogenic heterozygous variants in PSTPIP1 lead to excessive IL1 mediated inflammation, which is the cause of rare autoinflammatory syndromes: PAPA (pyogenic arthritis, pyoderma gangrenosum, acne) and the more recently described PAMI (PSTPIP1-associated myeloid-related proteinemia inflammatory). We investigated gene expression in patients reported in the following table to better understand the molecular pathogenesis of these diseases.

Methods

Transcriptomic studies of blood cells from our patients compared with a group of healthy subjects and pathway over-representation analysis to investigate the main biological processes involved.

Results

Transcriptomics analysis highlighted the heme metabolism pathway which is over-expressed among all the patients compared with controls, already known to be involved in physiological and pathological processes. Patients with PAMI showed neutrophils degranulation pathway over-represented, probably due to neutrophiles hyper-activation. Only pt5 presented a high-interferon-score that recovered after therapy with HCQ.

Conclusions

The presence of lupus-like manifestations in PAMI has never reported so far. Even if only one of our patients with PAMI displayed interferon-related inflammation, we can speculate a possible cross-talk between IL1 and interferon pathways. A possible hypothesis, which could be worth investigating, is that the increased neutrophil activation pathway found in PAMI could be associated to the release of NETs and to the activation of interferon signaling.

Background and Aims

Blau Syndrome (BS) is a very rare autosomal dominant inherited autoinflammatory disorder. Ophthalmic involvement is a severe, degenerative and often insufficiently controlled issue of BS. Currently, there are no predictive markers of disease at ocular level to target for treatments. Thus, we aim to investigate in depth the molecular aspects involved in ocular inflammation in BS.

Methods

To investigate the tear proteome of a BS patient with advanced eye degeneration, three healthy family members and three healthy controls, tear fluids collected via Schirmer’s strips were the starting materials. LC-MS/MS analysis was conducted with a LTQ-OrbitrapXL mass spectrometer. Data were searched against the Uniprot Database and filtered to consider only high confidence proteins. T test assessed the differential expressed proteins amongst samples.

Results

Overall, 291 proteins (145 differential expressed between BS and healthy subjects, 40 BS-associated, 1 control-associated) were identified and considered clinically interesting with a fold change >5. BS-associated overexpressed proteins included immunoglobulins and proteins related to the innate immunity and the proteolysis. Among the proteins mainly expressed in BS, alpha2-macroglobulin (increased 27 times) and SERPINA1 (increased 9 times) may have a role in sustained ocular inflammation. Of particular interest the finding of the under-expression of Phospholipase A2 in BS patient. This protein has a crucial role in inflammatory response and signal trasduction.

Conclusions

Our study provides a first insight into the ocular proteomic of BS. The newly described BS-associated proteins might become tools for eye degeneration risk assessment in BS patients.

Background and Aims

The low molecular weight fraction of human serum albumin (LMWF5A) is a biologic drug currently under clinical investigation for the treatment of osteoarthritis and the hyper-inflammation observed with severe COVID-19. In vitro investigations demonstrate that LMWF5A inhibits pro-inflammatory cytokine release (such as TNFα, IL1β, IL6, and CXCL10) as well as the activity of pro-inflammatory transcription factors (NF-κB and STAT) in immune cells stimulated through toll-like receptor (TLR) 4 using lipopolysaccharide. The aim of this investigation is to evaluate the ability of LMWF5A to suppress TLR7/8 signaling, which has been implicated in the pathology of a variety of inflammatory and autoimmune diseases including cytokine storm development and acute kidney injury in sepsis and lupus.

Methods

To evaluate this, human monocytic cell lines and peripheral blood mononuclear cells (PBMC) were activated in the presence of LMWF5A or saline using an agonist for TLR7/8 (CL075). CXCL10 release in the culture medium was then quantified by ELISA after 24 hours, and percent change was calculated versus TLR7/8-activated cells treated with saline as a diluent control.

Results

LMWF5A dose-dependently inhibited CL075-induced CXCL10 release from macrophage-like THP-1 cells from 15-90%. Furthermore, this drug response was observed across a range of CL075 concentrations and in a variety of monocytic lineages, including primary PBMC (54%) and U937 cells (82%).

Figure 1

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Conclusions

These findings provide evidence that LMWF5A treatment suppresses TLR7/8 signaling in monocytic/macrophage lineages and suggests a role for LMWF5A in treating the dysregulation of these pathways observed in lupus nephritis as well as in COVID-19.

Background and Aims

Myositis are chronic autoimmune diseases characterized by muscular weakness, inflammation, and extra-muscular involvement. Extracellular vesicles (EVs) are heterogeneous lipid bilayer nanoparticles, ranging from 30 nm to 1000 nm, naturally released from cells. Convoying their cargo, EVs allow intercellular communication. Their role in the pathogenesis of autoimmune diseases is recognized. The study aims to investigate EVs as potential myositis biomarkers.

Methods

EVs were isolated from platelet-free plasma of myositis patients and healthy donors (HDs) through size exclusion chromatography and enriched by ultrafiltration. They were observed by transmission electron microscopy (TEM) and quantify by tunable resistive pulse sensing (TRPS).

Results

By TEM, EVs were seen more concentrated in patients (n=4) than in HDs (n=5) samples. EVs appeared intact, roundish, and heterogeneous in size, with a prevalence of smaller EVs in both groups. TRPS measurements showed an EVs concentration of 1.76e+14 [EVs/mL] and a mode diameter of 80 nm, in the patient sample (n=1). The mean EVs concentration of HDs samples (n=2) was 1.12e+14 [EVs/mL] with a mean mode diameter of 77 nm (Figure 1).

Figure 1. TRPS measurement of EVs report (qNano, Izon instruments). Quantification of a myositis patient (on the left) and HD (on the right) samples. Particles distribution graphs show particles’ concentration [EVs/mL] related to their size.

Conclusions

Preliminary data based on TEM observation and TRPS measurements showed that EVs are predominantly small in both patient and HDs samples. Moreover, the EVs concentration appeared slightly higher in the patient rather than in HDs. Further analyzed employing classical and imaging flow cytometry are ongoing.

Background and Aims

Parathormone (PTH) has been clinically associated with autoimmune diseases but its pathophysiological impact has not been established. Our aim was to evaluate the expression of the PTH receptor (PTH1R) on B cells in patients with primary Sjögren’s Syndrome (pSS) and Systemic Lupus Erythematosus (SLE), and PTH effects on B cells in vitro.

Methods

We evaluated PTH1R expression on B cells and the receptor-expressing B cell subsets’ distribution (Bm1-Bm5) by flow cytometry in peripheral blood of SLE, pSS and healthy controls (HC). Analysis in vitro was performed in a B-cell line (Ramos) with PTH stimulus; effects on proliferation, apoptosis, and subpopulations were evaluated. We correlated PTH1R levels with ESSDAI and SLEDAI. Statistical analysis was performed using Kruskal Wallis and Spearman’s tests.

Results

We included 12 patients with SLE, 19 with pSS, and 20 HC. PTH1R expression on B-cells from SLE was 13% compared to pSS (4.4%), p=0,019; and HC (5%), p=0,013. Distribution of B cell subsets expressing PTH1R was significantly lower in Bm1 (23%), and higher in Bm2 (31.1%), and Bm2´ (6.4%) in pSS compared to HC (39%, 18%, 1.7%, respectively), p=0.010, 0.05, 0.028. Correlation between PTH1R levels and ESSDAI was r=0.511, p=0.026; SLEDAI was not correlated. Higher PTH concentrations tended to maintain B cells in Bm3-4 subset compared to unstimulated B cells that differentiated to eBm5 subset.

Conclusions

PTHR1 expression could be a useful biomarker in SLE and activity marker in pSS. PTH effects on B-cell subsets modulation suggest a potential role of the hormone in the pathophysiology of autoimmune diseases.

Background and Aims

Th17 and Tfh cells, sustain tissue inflammation and autoantibody production respectively in rheumatoid arthritis (RA). Tfh cells co-expressing Th17 markers (CXCR5+Th17), incorporate both pathogenic roles and are amplified in RA. We have previously shown that secukinumab, an anti-IL-17A biologic, decreases nuclear antigen-specific autoantibodies, plasmablasts and Tfh cells in patients with psoriatic disease (PSD). Our aim was to explore whether these cells can serve as predictive biomarkers of secukinumab-induced remission in PSD.

Methods

PBMCs were isolated from 12 psoriasis and 8 psoriatic arthritis (PsA) patients at baseline and 6-months following secukinumab administration. Disease activity and response to biological treatment was assessed using PASI and DAS28-CRP criteria respectively. Tfh cells, CXCR5+Th17 cells and plasmablasts were analyzed flow cytometrically using fluorochrome-conjugated monoclonal antibodies against conventional cell surface markers.

Results

8 out of 12 psoriasis and 4 out of 8 PsA patients were classified as complete responders (CRs). In the CR group, the frequency of CXCR5+Th17 cells (gated as CCR6+ cells within CXCR5+CD4+ cells), was significantly reduced at 6 months compared to baseline (p<0.01). In contrast, there was no significant difference between baseline and 6-month levels in the non-responder group (NR). The percentages of CXCR5+Th17 cells at baseline positively correlated with PASI and DAS28-CRP scores. Reduced percentages of CXCR5+Th17, Tfh cells and plasmablasts at 6 months (as compared to baseline levels) were associated with low PASI scores in psoriasis CRs.

Conclusions

High levels of CXCR5+Th17, Tfh cells and plasmablasts can be indicative of an impartial clinical response to secukinumab in psoriatic disease.

Background and Aims

Protein citrullination, key in Rheumatoid Arthritis (RA), is catalyzed by the protein-arginine deiminase (PAD) enzymes, which require calcium for their activity. These proteins can autocitrullinate but the impact on antibody recognition isn’t fully understood. The objective of this study was to evaluate the effect of PAD1 autocitrullination on the recognition by anti-PAD1 antibodies.

Methods

Full-length PAD1 proteins were recombinantly generated in the absence or presence of calcium. Citrullination status of the produced antigens was evaluated by immunoblotting using the anti-Citrulline (Modified) Detection Kit (MerckMillipore), alongside a commercial PAD1 protein (Cayman). A panel for the detection of anti-PAD1 IgG for the particle-based multi-analyte technology [PMAT, research use only (RUO), Inova Diagnostics] was created by conjugating the citrullinated and non-citrullinated PAD1 versions to magnetic beads. Sera from RA patients (n=14) and healthy controls (n=8) were tested with this panel on the Aptiva instrument (RUO, Inova Diagnostics).

Results

Autocitrullination of PAD1 antigens was confirmed by anti-citrulline immunoblot only for the proteins generated in the presence of calcium (Figure 1). Partial citrullination was observed for the commercial PAD1 antigen. Strong reactivity to both the citrullinated and non-citrullinated PAD1 antigens was observed with the PMAT anti-PAD1 IgG panel, with apparent higher reactivity to the non-citrullinated versions (Figure 2).

Conclusions

Both citrullinated and non-citrullinated PAD1 were recognized by anti-PAD1 IgG antibodies in the sera of RA patients, supporting the idea that these antibodies recognize unique epitopes in the PAD1 enzyme and are distinct from ACPA.

Background and Aims

B cells may be involved in inflammatory arthritis induced by checkpoint inhibitors (CPI-IA). Our aim was to investigate the phenotype of circulating B cells in patients receiving CPI treatment and the relationship with CPI-IA.

Methods

Peripheral blood mononuclear cells were analyzed with flow-cytometry in CPI-IA patients and in patients who had not developed immune-related adverse events upon CPI (non-irAE). B cells were identified as CD19⁺ and divided into naïve (CD27^-IgD⁺), memory (CD27⁺IgD^+/-) and transitionals (CD10⁺CD24⁺CD38^+/hi). Levels of the activation marker CD21 were also analyzed. Plasma levels of IL-6, IL-10 and BAFF were measured by ELISA. Non-parametric tests were used for comparisons.

Results

Six CPI-IA and 7 non-irAE patients matched for age, gender and CPI treatment were included. CPI-IA patients displayed increased levels of circulating B cells (p=0.002) and of transitional B cells vs. non-irAE (median %, range: 7.8 (4.5-11.4) vs. 3.2 (1.6-4.3), p=0.007), while no remarkable changes were seen across other subsets. Transitional B cells significantly decreased when CPI-IA resolved (p=0.008). Levels of CD21 on transitional B cells were increased in CPI-IA vs. non-irAE (p=0.01). There were no significant differences in cytokine levels between patient groups. IL-10 tended to increase while IL-6 tended to decrease at CPI-IA resolution, yet not reaching significance. BAFF levels significantly increased from active to quiescent CPI-IA (p=0.02).

Conclusions

Transitional B cells are expanded in CPI-IA patients. BAFF increase may parallel their decrease at CPI-IA resolution. Monitoring of transitional B cells could facilitate early diagnosis and treatment of CPI-IA.

Background and Aims

Hydroxychloroquine(HCQ) is a disease-modifying anti-rheumatic drug that is often used in patients with rheumatoid arthritis(RA), systemic lupus erythematosus(SLE), and other autoimmune diseases like Sjogren's Syndrome(SS) or Mixed Connective Tissue Disease(MCTD). It has been observed that in-vitro, SARS-Cov-2 viral replication is reduced by HCQ, but in doses higher than 1000mg. There is controversy about whether it can help in preventing or treating COVID-19, but it is not approved by any international association.

Our study aimed to evaluate and analyze the incidence of COVID-19 in patients with SLE, RA, SS and, MCTD who were taking Hydroxychloroquine for their rheumatic diseases.

Methods

This was a prospective study that included 145 patients with SLE, RA, SS, and MCTD who were on HCQ. The study began on 25/02/2020 (14 days before discovering the first cases of SARS-Cov2 in Albania) and was completed on 25 March 2021, coinciding with the start of massive vaccination. All patients were completed with SARS-Cov-2 serology and PCR.

Results

After analyzing the data, it was seen that in 24 patients(16.6%) were found positive IgG anti-Cov-2, 4 patients had experienced pulmonary complications, however, not requiring hospitalization. Twelve of them had RA, 3 had SS, 7 patients had SLE and 2 patients had MCTD. All IgG-positive patients had taken 200mg/day. The other 121 patients(83.4%) were found to have negative IgG anti-Cov-2 antibodies.

Conclusions

Our study resulted in a low incidence of COVID-19 in patients with rheumatic diseases on hydroxychloroquine. This suggests a protective role of this drug in rheumatic patients, even in moderate usual doses.

Background and Aims

With the rapid spread of the SARS-COV2 virus, the government of Singapore is pushing for mass vaccination.

During the last 2 months, we have been seeing an increasing number of patients with arthritis flares after the Pfizer BioTech COVID-19 vaccine. Vaccine-induced arthritis is part of the ASIA syndrome.

We report our experience with 22 patients who developed arthritis flares after the COVID-19 vaccination.

Methods

We collated all our patients who developed inflammatory arthritis after COVID-19 vaccination from March to May 2021.

The patients were selected if they developed arthritis after the first or second dose of the vaccine within 4 weeks of the dosing, and the patients had no previous arthritis history or were in remission or stable disease for at least 3 months prior to receiving the COVID-19 vaccination.

Results

Racial distribution: 18 Chinese, 2 Indian, 1 Filipino 1 British..

Age ranged from 45-85 years, mean age of 59 years.

Diagnosis: Rheumatoid arthritis (RA) flare – 10, Osteoarthritis flare – 5, Gouty arthritis flare -4, Pseudogout flare - 1 De novo oligoarthritis – 2.

Flare Characteristics::Mild – 4, Moderate – 12, Severe – 6

Monoarticular – 12 Oligoarticular – 4 Polyarticular – 4

Flare after 1st dose: 10 Flare after 2nd dose: 10 Recurrent Flare after Both doses: 2

Time to Onset of flares: 1- 27 days.

Conclusions

With widespread COVID-19 vaccination, ours is the largest series of series of post-vaccination rthritis flares. The BNT162b2 mRNA vaccine seems to reprogram both the body’s adaptative and innate immune responses, More observational studies are needed.