Welcome to the 13th International Congress on Autoimmunity interactive program

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Autoantibodies are important parameters in the diagnosis of autoimmune diseases. Since the discovery of the first autoantibodies, many researchers and laboratories aimed to measure multiple antibodies simultaneously which led to the invention of several unique methods. During early days, countelectrophoresis became a popular tool followed by the introduction of westernblots and line immunoassays. Later on, planar and particle-based arrays were developed and deployed for multi-analyte testing as an integral part of autoimmune research and diagnostics. Although it seems intuitive to test multiple antibodies simultaneously, multiplex assays are not without challenges and drawbacks which include the pre-test probability and reimbursement limitations as well as technical roadblocks. Consequently, fully automated, random access, single analyte tests were developed and deployed to autoantibody testing. Due to the need for patient stratification in autoimmune diseases, the demand for more efficient and highly reliable multi-analyte assays continues. Here we provide a historical journey and important aspects of autoantibody profiling as part of autoimmune research and diagnostics.

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Antibodies against the fibrillar cartilage collagen type II (anti-CII) can be detected in 6-9% of patients with rheumatoid arthritis (RA). Whereas rheumatoid factor and antibodies against citrullinated peptides/proteins (ACPA) can be detected many years before RA onset, anti-CII are absent during the years predating clinical disease, but seem to appear close to diagnosis in conjunction to clinical symtoms. We have described that in anti-CII positive patients, autoantibody levels are high at the time of diagnosis and thereafter decrease during the first year. We have developed an in vitro model to study the functional effects of surface-bound anti-CII-containing immune complexes (IC), mimicking how anti-CII can act when binding to CII exposed in hyaline cartilage in RA joints. Using that model, we have shown how anti-CII-containing IC induce the inflammatory cytokines TNF-α, IL-1β and IL-8 via FcγRIIa-mediated macrophage stimulation, and that granulocytes are especially important by co-stimulating macrophages to produce IL-8 and other chemokines via a mechanism dependent on TLR4 and the granulocyte enzymes elastase, cathepsin and myeloperoxidase. In agreement with these proinflammatory functions in vitro and declining anti-CII levels in vivo after RA diagnosis, anti-CII positive patients show an RA phenotype with acute onset with high levels of inflammatory markers but with good long-term prognosis This represents the opposite to ACPA-positive RA where the long-term prognosis is poor. Thus, analysis of anti-CII might be a means to define a group of newly diagnosed RA patients where special therapeutic considerations might be applicable. This hypothesis is currently investigated in large RA cohorts in an ongoing Scandinavian collaboration.

In this talk I will present our work on the functional role of anti-CII in vitro as well as our clinical studies in propectively followed RA cohorts.

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TSPAN32 is a member of the tetraspanin family, which is actively involved in the regulation of the immune responses. Previous studies have investigated the role of TSPAN32 in Multiple Sclerosis and Systemic Lupus Erythematosus, however no data are currently available about its role in Rheumatoid Arthritis (RA). In the present study, we have evaluated the expression levels of TSPAN32 in immune cells from a spontaneous mouse model of RA, the IL1ra knockout (KO) mouse, as well as in circulating and synovial-infiltrating immune cells from RA patients. TSPAN32 expression resulted significantly reduced in splenic CD4+ T cells from IL1ra KO mice, at 1 and 4 months of age. On the other hand, no significant modulation was observed for the other members of the tetraspanin family, with the exception of TSPAN4 and TSPAN31, which were significantly increased as compared to the wild-type mice. Along the same lines, a significant reduction in TSPAN32 expression was observed in CD4+ effector memory T cells from RA patients, as compared to healthy donors, and also a moderate but significant downregulation of TSPAN32 was observed in the inflammatory HLA-DR+CD27- cells from RA patients, as compared to those from OA patients. Overall, our data suggest that the reduced expression of TSPAN32 characterizes the pathogenetic T cell populations in RA, and that this may contribute to trigger the arthritogenic immune responses. Furthermore, our results strengthen the hypothesis that defective TSPAN32 expression may represent an important immunopathogenetic abnormality that could play a role in the pathogenesis of Th1/Th17-driven disorders.

Background and Aims

Rheumatoid arthritis (RA) is a systemic autoimmune disease, caused by the combination of genetic factors, environmental factors, and a role for gut microbiome is also suspected at preclinical stage. However, how these factors interact remains unclear, which is the aim of this study.

Methods

54 patients with RA, 48 preclinical individuals including 8 who have developed to RA and 31 controls (HC) were selected from the Tatarstan’s cohort, and tested for gut microbiota (16S rRNA). The analysis of RA-associated factor included HLA locus variability (HLA-A/B/C/DQ and DRB1), cytokine polymorphism, haplomitotype determination, and a large panel of environmental factors.

Results

No difference was observed when considering alpha diversity, beta diversity, and microbiome composition between samples from controls, preclinical individuals and RA patients. In contrast HLA-DRB1 (SE, beta diversity) and the education level (alpha & beta diversity) influence microbiome. At genus level, Prevotella species including Prevotella copri, were more abundant in controls and at preclinical stage within the low education subgroup. Next, an immunization against Prevotella sp is suspected as a reduction was observed among RA and preclinical individuals evolving to RA among the low education/HLA-DRB1-SE subgroup.

Conclusions

Prevotella sp is overexpanded in stool samples from low educated individuals and among then HLA-DRB1-SE represents a risk factor for RA.

Funding: “Russian Foundation for Basic Research” (№ 20-515-05006)

Background and Aims

Finding out which rheumatoid arthritis (RA) patients will show a poor response to first-line therapy is currently based on trial and error. The aim of this study was to find novel antibody biomarkers that identify at baseline, which RA patients fail to reach remission, or low disease activity (LDA), after intensive classical synthetic disease-modifying anti-rheumatic drug (csDMARD) therapy.

Methods

cDNA phage display libraries expressing RA synovial antigens were screened for antibody reactivity in baseline sera from participants of the CareRA trial failing to reach early DAS28CRP remission (rem-) after intensive csDMARD therapy. Presence of antibodies to the identified University Hasselt (UH)-RA antigens was validated using ELISA in 179 baseline CareRA samples, and correlated with different criteria for remission or LDA during follow-up.

Results

Baseline antibody reactivity against 3 antigens, UH-RA.305, UH-RA.318 and UH-RA.329, was significantly higher in patients failing to reach DAS28CRP or DAS28ESR remission, or SDAI or CDAI LDA at week 8, compared to RA patients reaching these criteria. Baseline anti-UH-RA.305/318/329 antibody reactivity correlated with lack of DAS28CRP remission at week 8, 16, 40 and 52, and was present in one in three DAS28CRP rem- patients, and in one in two DAS28CRP rem- RF/ACPA seronegative patients. For each of the remission criteria studied, baseline antibody reactivity against the UH-RA.305/318/329 antigens was significantly higher in patients that did not maintain sustained disease remission, or sustained LDA during the first year.

Conclusions

We identified 3 antibody biomarkers that predict the absence of response to first-line RA therapy and could contribute to personalized medicine decisions.

Background and Aims

With a prevalence of approximately 1%, rheumatoid arthritis (RA) is one of the most frequent autoimmune disorders. Treatment at early stages can significantly slow down disease progression or even prevent irreversible damages. However, an early diagnosis is often challenging. Up to 50% of early RA patients are negative for the standard serological markers rheumatoid factor (RF) or anti-cyclic citrullinated peptide (CCP) antibodies. A more comprehensive analysis of antigenic proteins and their underlying epitopes can provide the necessary information for innovative serological tests with higher sensitivity for early diagnosis of RA.

Methods

To identify novel biomarkers, we applied high-density peptide microarrays displaying large numbers of autoantigens/antigen candidates converted into >100.000 overlapping peptides including all citrullinated and carbamylated variants. Using this highly diverse library, we screened sera from different disease and control cohorts and identified various epitopes with a higher prevalence in previously seronegative early-stage patients.

Results

Bioinformatic analysis combined with machine learning resulted in a peptide biomarker combination, which allowed the diagnosis of early seronegative RA vs. healthy controls with an accuracy of 87% and early seropositive RA vs. healthy controls with an accuracy of 94%, respectively. The validation of the marker set using well-characterized patient samples (n=1027) by Aptiva particle-based multi-analyte technology (Werfen) revealed highly significant peptides for discriminating RA vs. controls. Logistic regression modeling demonstrated a higher sensitivity but slightly lower specificity when analyzing CCP3 in combination with the marker set as compared to CCP3 alone.

Conclusions

Hence, the novel marker set has the potential to improve the early diagnosis of RA.

Background and Aims

Systemic sclerosis (SSc) is characterized by progressive cutaneous and organ fibrosis. Antibody profiles can aid in diagnosis and identification of patterns associated with clinical progression. While SSc occurs worldwide, the prevalence of specific antibodies appears to differ between western and eastern populations. Our aim was to compare autoantibody profiles obtained by novel particle-based multi-analyte technology (PMAT) assays on SSc patients from China and Europe.

Methods

Autoantibodies to 20 autoantigens were assessed in sera from 266 Chinese (Fudan University, Shanghai) and 198 Italian (Policlinico Hospital,Milan) SSc patients by PMAT. 223 Chinese and 158 Italian patients had classification criteria data available [(diffuse vs. limited cutaneous SSc (dcSSCA/lcSSc)].

Results

The ratio between dcSSc vs. lcSSc was 39.6 vs.:60.1% (China) and 16.0 vs. 71.7% (Italy) (p <0.0001). Notable differences observed between the two cohorts included higher prevalence of antibodies to RNP (22.0 vs. 7.6%), Scl-70 (50.2 vs. 35.4%), Ro52 (26.0 vs. 16.5%), Ro60 (44.7 vs. 18.4%) and lower ACA (14.8 vs. 48.7%), BICD2 (1.3 vs. 13.3%), PM/Scl (1.8 vs. 11.4%) prevalence in Chinese cohort.

Conclusions

Our study supports previous reports of higher prevalence of antibodies to RNP, Scl-70, and lower prevalence of ACA in Chinese SSc patients and adds new data on the prevalence of novel biomarkers such as BICD2, which was rarely detected in Chinese patients in contrast to European patients. Detailed autoantibody profiles easily obtained from PMAT analysis provides a foundation for potential optimization of clinical management and treatment of SSc patients based on individual autoantibody profiles. Our preliminary data merits further verification.

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Patients with ASIA present with complaints such as fatigue, cognitive impairment, arthralgias, myalgias, pyrexia, dry eyes, and dry mouth. During the last decennium, it has been postulated that these symptoms in patients with foreign body implants are due to a chronic inflammatory process and an adjuvant effect of the implanted biomaterial. Ultimately, these inflammatory reactions result in (an increase of) allergies, autoimmune diseases, immune deficiency, and/or lymphomas. Pre-existent allergic disease has been found to be an important risk factor for the development of ASIA after foreign body implantation. Explantation of the foreign body results in the majority of patients in an amelioration of the symptoms.

The best-studied example of foreign body implantation that may result in ASIA is silicone-breast implantation (SBI). Previously, we calculated from the literature that explantation of the SBI improved silicone-related complaints in 75 % of the patients (469 of 622). In patients with autoimmune diseases, however, additional (immunosuppressive) therapy is needed. There is some evidence that explantation with total capsulectomy may result in better results than explantation without capsulectomy, although good prospective studies are lacking. Recently, thousands of patients have been described demonstrating that SBI explantation results in an amelioration of ASIA symptoms. Several possibilities which could clarify why patients improve after explantation have been postulated. Firstly, the inflammatory response could be reduced after explantation, supporting a causal role for SBI in the pathogenesis of ASIA. Secondly, explantation of the implants may remove a nociceptive stimulus, which may be a causative factor for many complaints as well. Recently, studies have been published that not only suggest recovery of symptoms but also improvement of objective measures after explantation, suggesting that SBI indeed causes ASIA. Options for the reconstruction of the explanted breast such as autologous tissue and/or the use of saline- or hydrocellulose-filled breast implants will be discussed.

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To date, around 60% of the world population has been protected by vaccines against SARS-CoV-2, signifcantly reducing the devastating efect of the pandemic and restoring social economic activity through mass vaccination. Multiple studies have demonstrated the efectiveness and safety of vaccines against COVID-19 in healthy populations, in people with risk factors, in people with or without SARS-CoV-2 infection, and in immunocompromised people. According to the criteria for post-vaccine adverse events established by the World Health Organization, a minority of individuals may develop adverse events, including autoimmune syndromes. The exact mechanisms for the development of these autoimmune syndromes are under study, and to date, a cause-efect relationship has not been established. Many of these autoimmune syndromes meet sufcient criteria for the diagnosis of Adjuvant-Induced Autoimmune Syndrome (ASIA syndrome). The descriptions of these autoimmune syndromes open new perspectives to the knowledge of the complex relationship between the host, its immune system, with the new vaccines and the development of new-onset autoimmune syndromes. Fortunately, most of these autoimmune syndromes are easily controlled with steroids and other immunomodulatory medications and are short-lived. Rheumatologists must be alert to the development of these autoimmune syndromes, and investigate the relationship between autoimmune/infammatory symptoms and vaccination time, and assess their therapeutic response.

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A clear association between the clinical picture of symptomatic women with silicone breast implants and dysregulated immunity was in dispute for decades. Recently, our group found, in a large-population based study, that women with silicone breast implant (SBI) have a significant increased risk to develop autoimmune diseases. Moreover, we reported a significant change in the titters of circulating IgG autoantibodies directed against G-protein coupled receptors (GPCRs) of the autonomic nervous system in symptomatic women with SBI, which might explain some of their subjective and autonomic-related manifestations. So far, the potential pathogenic functional effects of autoantibodies derived from symptomatic women with SBI have not been explored. In the current study, we describe for the first time, the in vitro and in-vivo functional activity of purified IgG antibodies derived from symptomatic women with SBI. Our in vitro studies shows that while healthy-derived IgG reduces the secretion of pro-inflammatory cytokines (TNFα and IL-6) by human monocytes in response to bacterial lipopolysaccharide (LPS), IgG derived from SBI women increases the production of these cytokines. Importantly, we directly injected IgGs derived from symptomatic women with SBI (suffering from autonomic-related manifestations such as cognitive impairment and depression) into mice brains. Behavioral studies demonstrated a specific and transient significant increment (about 60%) in the time spent at the center of the open field arena as compared with mice injected with IgG from healthy women (without SBI), apparently indicating anxiolytic outcome of SBI-IgG. This effect was accompanied with a strong trend of reduction of the locomotor activity of the SBI-IgG treated mice, indicating an apathic behavior masked by an anxiolytic-like behavior. Our study is the first to show the pathogenic functional activity of IgG autoantibodies in symptomatic women with SBI, emphasizing the importance of these antibodies as potential targets for antibody-depleting therapies in silicone breast implant related illness.

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Abstract.
Vaccination is one of the most successful public health programs in the history of medicine. As result of global immunization campaigns, several dreaded diseases have been eradicated. Nevertheless, modern excessive vaccination with persistent immune system hyperstimulation can theoretically lead to the development of autoimmune illnesses in predisposed individuals. Unfortunately, vaccine safety has become a medical dogma; vaccine adverse event reports are often shunned as anti-vax propaganda.

Independent clinicians from different parts of the world have described the onset of a chronic debilitating illness soon after HPV vaccination. This illness is characterized by headache, fatigue, widespread pain, dizziness nausea and abnormal limb movements, among other vexing symptoms. This constellation of symptoms and signs has been labeled with different diagnoses such as complex regional pain syndrome, postural orthostatic tachycardia syndrome, myalgic encephalomyelitis/chronic fatigue syndrome, or fibromyalgia. Profound dysautonomia and small fiber neuropathy have been reported in these cases.

This purported Post-HPV vaccination syndrome resemble other contested vaccine-related illnesses such as Macrophagic Myofasciitis and Gulf War Illness. We propose that vaccine-triggered, immune-mediated autonomic dysfunction and small fiber neuropathy could lead to the development of these post- vaccination syndromes in genetically susceptible individuals. Dorsal root ganglia may play a key role in the pathogenesis of these syndromes. These para-vertebral ganglia house the pain-transmitting small nerve fiber nuclei, each individual nucleus is enveloped by immune-competent glial cells. Lymphocytes, macrophages, and different pro-nociceptive mediators populate DRG. Communicating nerves tightly link DRG with the para-vertebral sympathetic chain. Dorsal root ganglia are able to actively trapped antigen specific antibodies including vaccines. We proposed that in susceptible individuals vaccines may be trapped at the dorsal root ganglia inducing inflammation, dysautonomia and small fiber neuropathy.