Explore the vast range of titles available.

Autoimmune diseases (AIDs) are complex diseases characterized by persistent or recurrent inflammation, alteration of immune response, and production of specific autoantibodies. It is known that different AIDs share several susceptibility genetic loci. Tumor necrosis factor alpha inducible protein 3 (TNFAIP3) encodes the ubiquitin-modifying enzyme A20, which downregulates inflammation by restricting NF-κB, a transcription factor that regulates expression of various proinflammatory genes. Variants in TNFAIP3 gene have been described as associated with susceptibility to several AIDs. Here, we analyzed two TNFAIP3 polymorphisms in Italian patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and primary Sjogren’s syndrome (pSS), to verify if the genetic variability of TNFAIP3 gene is involved in genetic predisposition to AIDs also in the Italian population. We recruited 313 SLE patients, 256 RA patients, 195 pSS patients, and 236 healthy controls. Genotyping of rs2230926 and rs6920220 in TNFAIP3 gene was performed by an allelic discrimination assay. We carried out a case/control association study and a genotype/phenotype correlation analysis. A higher risk to develop SLE was observed for rs2230926 (P=0.02, OR=1.92). No association was observed between this SNP and the susceptibility to pSS or RA. However, the rs2230926 variant allele seems to confer a higher risk to develop lymphoma in pSS patients, while in RA patients, the presence of RF resulted significantly associated with the variant allele. Regarding the rs6920220 SNP, we observed a significant association of the variant allele with SLE (P=0.03, OR=1.53), pSS (P=0.016, OR=1.69), and RA (P=0.0001, OR=2.35) susceptibility. Furthermore, SLE patients carrying the variant allele showed a higher risk to develop pericarditis, pleurisy, and kidney complications. Our results support the importance of the TNFAIP3 gene variant role in the development of different autoimmune diseases in the Italian population and furtherly confirm a sharing of genetic predisposing factors among these three pathologies.

Background:To fulfil the energy demands upon activation, lymphocytes undergo a profound metabolic reprogramming (i.e switch towards glycolysis), which sustains their pro-inflammatory function. This concept, which is known under the term “immune-metabolism” [1], has become of great interest in rheumatic diseases; however, little is known in Sjogren Disease (SD). As infiltrating T and B cells forming aggregates (named foci) are the hallmark of SD and display a major pathogenic role, investigating the contribute of metabolic reprogramming, especially glycolysis, in these cells looks particularly relevant.Objectives:Aim of this study is to evaluate the expression of the glucose transporter marker GLUT-1 in lymphocytes infiltrating SD minor SG and to assess its correlation with the severity of inflammatory infiltrates.Methods:The expression of GLUT-1 on CD3+ and CD20+ lymphocytes infiltrating minor SG from patients with SD, has been evaluated by IFI in 9 different biopsies samples. Biopsies were divided in three groups according to composition and the level of organization of the foci:-G1: small foci mainly composed by CD3+ cells (Small) (n=3)-G2: large foci segregated in CD3+ and CD20+ areas [Segregated Foci (SF)] (n=3)-G3: large SF with CD21+ staining indicative of Germinal Center like structures (GC) (n=3)The quantification of the expression of GLUT-1 on lymphocytes was performed by digital imaging analysis (DIA) with QuPath software. GLUT-1 expression was quantified overall and selectively on CD3+ or CD20+ lymphocytes. To visualize SG areas with the highest expression of GLUT-1, density-maps were generated. Analysis of variance (ANOVA) and pairwise comparisons (Bonferroni correction) were performed to evaluate differences in the expression of GLUT-1 both according to the grading of inflammation (G1-G2-G3) and according to the type of foci (Small-SF-GC).Results:Across the three groups (G1-G2-G3), a remarkably different GLUT-1 expression on infiltrating lymphocytes was detected (Figure 1a). Despite the evident increase from G1 to both G2 and G3, statistical significance was not achieved in pairwise comparisons [G1 (9,6%±3,8) vs G2 (37,7%±13,7), p=0.069; G2 vs G3 (34,1%±3,7), p=1]. The selective analysis for CD3+ and CD20+ cells showed similar results. Compared to G3, in G1 and G2 CD20+ cells showed a slightly lower rate of GLUT-1 (Figure 1a). Across the three types of foci, GLUT-1 expression on lymphocytes also significantly differed (Figure 1b). In pairwise comparison a significant difference between Small (13,7%±11,8) and SF (31,7%±17,9)(p=0.008) was detected while significancy was not reached in Small vs GC (26,8%±11,7)(p=0.338) and SF vs GC (p=1) (Figure 1b). Similar results were obtained when CD3+ and CD20+ cells were analysed separately (Figure 1b). Representative images of GLUT-1 density-maps in the G3 group and GLUT-1 expression in the different types of infiltrates are reported in Figure 1c-d.Conclusion:This is the first study identifying the expression of the glycolysis marker GLUT-1 on both T and B lymphocytes infiltrating SD SG. An increase in GLUT-1+ lymphocytes was detected according to the severity of inflammation and reached the highest levels in large, organized foci. Despite preliminary, our findings suggest that activation of glucose metabolism may be associated with tissue lymphocytes activation and proliferation. Validation studies on a larger sample size are ongoing to confirm these findings and further investigate the role of glycolysis in SD infiltrating lymphocytes.REFERENCES:[1] O’Neill LA, Kishton RJ, Rathmell J. A guide to immunometabolism for immunologists. Nat Rev Immunol. 2016 Sep;16(9):553-65Figure 1.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:Systemic sclerosis (SSc) is characterized by progressive fibrosis and microvascular dysfunction that involves multiple organ systems, including kidneys. Kidney involvement, beyond the scleroderma renal crisis (SRC), is often asymptomatic and underdiagnosed, associated with subclinical renal vasculopathy characterized by abnormalities in renal microcirculation and mild changes of glomerular filtration rate (GFR). Kidney involvement is rare in isolated Sjögren’s syndrome (SS) and reported between 5% and 14% in european patients and approximately 30% in asian patients.Objectives:The aim of the study was to assess renal involvement in SSc and SS and follow its progression over a three-year period.Methods:Patients with SSc (2013 EULAR criteria) and isolated SS (2016 ACR/EULAR criteria) were consecutively enrolled. Demographic and clinical characteristics at baseline (T0) were gathered including sex, age, body mass index (BMI), history of essential hypertension and diabetes, serum creatinine, and estimated GFR calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. Exclusion criteria were presence of SRC, atherosclerosis renal artery stenosis and other secondary causes of hypertension, glomerulonephritis, urinary tract obstruction, urinary infections, heart failure, pulmonary arterial hypertension, malignancies. All patients were followed for three years (T1) with renal function assessment. Group comparisons were made using the Student’s t-test or Mann-Whitney test for continuous variables and the chi-square test or Fisher’s exact test for categorical variables. A significance level of p<0.05 was considered.Results:A total of 248 patients were enrolled, 127 SSc and 121 SS patients. Their features at baseline (T0) are summarized in Table 1. Table 2 summarizes the comparative analysis of renal function between SSc and SS patients at baseline and after 3 years. SS patients had similar median serum creatinine both at T0 and at T1 [0.78 mg/dl (IQR 0.68;0.84) vs 0.78 mg/dl (IQR 0.7;0.84), p>0.05], but a higher eGFR at T0 than at T1 [87.9 ml/min (IQR 72.6;102.3) vs 86.4 ml/min (IQR 72.7;96.7), p<0.05], with a variation of −2.1 ml/min (IQR −11.4;1.1). SSc patients had a statistically significant lower median serum creatinine at T0 than at T1 [0.7 mg/dl (IQR 0.6;0.8) vs 0.8 mg/dl (IQR 0.7;0.9), p<0.001], with a variation of 0.1 mg/dl (IQR 0;0.2), and a statistically significant higher median eGFR at T0 than at T1 [97 ml/min (IQR 85;108.5) vs 91 ml/min (IQR 73;103), p<0.001], with a variation of −3 ml/min (IQR −18;−1). In both SSc and SS eGFR median variation was statistically significant higher in patients affected by systemic arterial hypertension compared to those without [−12.2 ml/min (IQR −16.32;5.42) vs −1.8 ml/min (IQR −3;0.4), p<0.01 for SS patients and −18 ml/min (IQR −26; −12) vs −2 ml/min (IQR −7;2.25), p<0.001 for SSc].SSc patients had a significantly higher variation compared to SS patients both for median serum creatinine [0.1 mg/dl (IQR 0;0.2) vs 0 mg/dl (IQR −0.1;0.1), p<0.05] and median eGFR [−3 ml/min (IQR −18;−1) vs −2.1 ml/min (IQR −11.4;1.1), p<0.05].Conclusion:In both SSc and SS patients, renal involvement exhibited a subclinical pattern (eGFR > 60 ml/min). This preliminary study also highlights slight differences in renal involvement and its progression between the two groups considered. In SSc patients, creatinine levels at T0 are, on average, lower than in pSS patients possibly linked to their reduced muscle mass. Over time, SSc patients tend to show a tendency toward eGFR reduction, for the continuous microvascular damage leading to chronic hypoxic-ischemic injury.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Background:In Sjögren Disease (SD) salivary gland epithelial cells (SGEC) sustain inflammation due to their acquired capacity to express/secrete adhesion/pro-inflammatory molecules. However, the mechanisms responsible for SGECs activation remain undetermined. The link between immune cells’ function and cells’ metabolic reprogramming is known under the term “immunometabolism” and has become one of the most exciting areas of investigation in the field of immuno-rheumatology, despite its relevance in SD has not been investigated as yet.We hypothesize that the altered cell energy metabolism of SGECs is a central driver of SG inflammation in SD and one that is targetable for the development of innovative therapies.Objectives:I–To determine (in vivo) in a viral-induced model of experimental sialoadenitis the relationship between SG inflammation and cell metabolic reprogramming;II–To identify ex vivo the main metabolic pathways differing between SD and sicca (control) SGECs;III–To assess ex vivo and in vitro the expression of glycolysis in SGECs and to investigate its contribution to SGECs activation.Methods:Aim I- SD mouse models were produced. After culling at different time points [day_0 (non-cannulated), day_5-12-19 (cannulated)], SG bulk RNA-seq analysis focused on metabolic pathways was conducted. Aim II- The metabolic profile of cultured SGECs from SD and sicca patients was analysed (mass spectrometry). Aim III- The expression of the glycolysis marker GLUT-1, along with lymphocytes (CD45+) and epithelial cells (PanCK+, CK7+, CK14+) markers, was determined (IFI) on paraffin-embedded sections of SD and sicca minor SG. SGECs cultures were treated as follows: 1) fluorescent glucose (6-NBDG), to assess glucose up-take; 2) 2-deoxy-glucose (2DG), to evaluate the effect of glycolysis inhibition on SGECs activation [ICAM-1 expression (IFI) and IL-6/type-I IFN production (Elisa and IFNα/β reporter cells)]; treatment conditions are in Figure 1d.Results:Aim-I: Following the inflammatory insult, bulk RNA-seq analysis in AdV-induced sialoadenitis, demonstrated changes in the SG metabolic profile. Down-regulation of specific pathways (i.e. oxidative phosphorylation and pyruvate metabolism), occurred up to day 12 post-cannulation (Figure 1a).Aim-II: A metabolic difference between SGECs from SD and sicca patients was revealed (i.e. glycolysis and TCA cycles) (Figure 1b).Aim III: As glycolysis turned out to be one of the most expressed pathways in SD, we further investigated its expression in SGECs. The expression of the glucose transporter GLUT-1 was highly detected in SD SG tissue by SGECs wile no expression was detected in sicca. Such GLUT-1 expression was mainly localized in ducts, especialy in ductal luminal cytokeratin-7 positive (CK7+) cells (Figure 1c). In vitro treatment of SGECs with fluorescent glucose demonstrated an higher glucose uptake in SD SGECs (representative example-Figure 1c). In primary SGECs culture, following stimulation with Poly(I:C), which mimics viral sensing by SGEC, an increase in IL-6, type-I IFN and the adhesion molecule ICAM-1 was observed. SGECs treatment with the glycolisis-inhibitor 2DG prevented [if Poly(I:C) and 2DG were administered concomitantly] or reduced [if 2DG was added following 12h stimulation with Poly(I:C)] the expression of adhesion/pro-inflammatory molecules (Figure 1d).Conclusion:In vivo experiments confirm that inflammation is intimately linked with metabolic changes occurring in SD SG. After induction of inflammation, we observed a down-regulation of oxidative phosphorylation and changes in glucose metabolism. Mass spectometry analysis confirmed the higher expression of specific metabolic pathways in SD SGECs, such as glycolysis. The increased expression of GLUT-1 in SD CK7+ ductal epithelial cells and the higher glucose up-take in SD cultured SGECs confirm such a shift towards glycolysis. Remarkably, glycolysis inhibition affects SGECs activation, indicating that metabolic reprogramming is likely responsible for their pathogenic behaviour. In vivo investigations on the potential utility of drugs targeting glycolysis in SD are currently ongoing.REFERENCES:NIL.Figure 1.Acknowledgements:The presenting author of this abstract (SC) is recipient of a “Career Research Grant” funded by FOREUM – Foundation for Research in Rheumatology.Disclosure of Interests:None declared.

Background:Sjögren syndrome (SS) is an autoimmune disorder characterized by chronic inflammation of exocrine glands and histological features of focal lymphocytic sialoadenitis. While the innate immune mechanisms display a major role at early stages of the disease, the adaptive immune system is the main driver of inflammation at later phases. Although the innate immune pathway of IL-1 displays great pro-inflammatory effect in different inflammatory conditions, its role in SS is still poorly defined. The so called ”activation” of salivary gland epithelial cells (SGECs) is key in the pathogenesis of SS and we recently described how this process is intrinsically linked to the activation of autophagy. The mechanisms behind SGECs activation remain unclear as well as their relationship with innate immune pathways and, in particular, with the IL-1 pathway. The few available data suggest that the expression of IL-1β is upregulated in activated epithelial cells from SS lacrimal glands; accordingly, increased assembly of the inflammasome complex NLRP3 has been described in the SG of SS mice models. Little is known about the role of the other isoform of IL-1 named IL-1α, an “alarmin” passively released by cells following damage with interesting effects in different rheumatic conditions.Objectives:Aim of this study is to investigate the IL-1 pathway, with particular regard to the expression of the two isoforms of IL-1 (α and β), in SS salivary glands and to assess its role in SGECs activation and homeostasis.Methods:The study was divided in two parts, a descriptive and a functional one. Descriptive study – Paraffin embedded minor SG biopsies (MSG) were collected from patients with SS and sicca syndrome (controls). Immunofluorescence staining for IL-1α and IL-1β detection were performed along with markers to localize their expression; these markers included SGECs markers (panCK, CK7, CK14) and lymphocytes markers (CD45, CD3, CD20). Functional study – SGECs cultures from SS and sicca MSG were produced. Supernatants were analysed by ELISA for the detection of IL-1 family cytokines. On a human SG cell line (HSG), in vitro treatments with activating stimuli [Poly(I:C) and LPS] and then with IL-1 inhibitors (Anakinra, blocking both IL-1α and IL-1β; Canakinumab, selectively binding IL-1β) were performed. Following treatments, changes in the expression of autophagy (LC3IIB and p62), apoptosis (annexin V) and activation (ICAM-1) markers were assessed by Western Blot and flow cytometry.Results:Descriptive study - IL-1α expression was detected both in MSG sections from patients with SS and in MSG from patients with sicca; its expression was mainly localized in luminal CK7 positive ductal epithelial cells. No expression was detected in acinar cells and infiltrates. The expression of IL-1β was not detectable neither in infiltrates nor in SGECs (Figure 1a). Functional study - Analyses of supernatants from SGECs cultures revealed slightly higher levels of IL-1α and IL-1β in SS compared to sicca, however the difference was not statistically significant (Figure 1b). Following HSG stimulation with Poly(I:C) and LPS, an increase in autophagy (increased LC3IIB, decreased p62) and activation (ICAM-1), and a slight increase in apoptosis (annexin V), were observed. Both treatments with Anakinra and Canakinumab induced a reduction in autophagy and a slight downregulation of activation markers; only anakinra determined a decrease in apoptosis while canakinumab displayed opposite effects (Figure 1c, d).Conclusion:The lack of expression of IL1β at tissue level might suggest that this pathway is mainly involved in the early phases of the disease rather than during the course of an established disease. Despite the absence of specificity for SS, the expression of IL1α at ductal epithelial cell level is quite interesting and worth of further investigation. In addition, the indirect evidence of an alteration in homeostasis and activation of SGECs following inhibition of IL-1 pathways, might suggest a role of this molecule in two of the major pathogenic mechanisms driving the development of an autoimmune epithelitis.Figure 1.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

BackgroundPolymyalgia rheumatica (PMR) is a chronic inflammatory disease that affects individuals of 50 or more years of age. It is characterised by shoulder and pelvic girdle pain, morning stiffness, functional limitation, and an increase in inflammatory indexes. Glucocorticoids (GC) represent the cornerstone of treatment. The pathogenesis is still not completely understood, although many shreds of evidence show the role of innate immunity.ObjectivesTo evaluate whether peripheral blood mononuclear cells (PBMCs) of patients with newly diagnosed PMR and not yet treated with GC show a baseline activation that is affected by treatment with GC.MethodsWe carried out an observational study, enrolling patients with PMR (2012 EULAR/ACR criteria) not yet treated with GC and controls. Blood samples were obtained from patients at baseline (T0) and after 4 months since the beginning of GC (T4), and from controls. An ultrasound (US) of shoulder and pelvic girdles was performed in PMR patients at T0 and T4. Demographic, clinical and US results were recorded in an electronic database. Patients with a personal history of rheumatic, inflammatory or oncologic diseases or subjects that received such a diagnosis during the study were excluded. PBMCs were isolated by density gradient centrifugation and analyzed in triplicates, among which one was stimulated with lipopolysaccharide (LPS). Then, the supernatant was collected and ELISA was performed for the determination of IL-1β, TNF, and IL-6 levels. The same cytokines were searched in the sera of PMR patients at T0 and at T4 and in the controls.ResultsWe consecutively enrolled 8 patients with a new diagnosis of PMR (F/M 6/2; median age/IQR 73/9.75 years; median disease duration 2/2.75 months) and 8 individuals without PMR matched for gender, age, and comorbidities with PMR patients (F/M 6/2; median age/IQR 69/10.5 years). We found higher concentrations of IL-1β and IL-6 in the unstimulated supernatant of PMR patients at T0 compared to T4 (p=0.0313 for both) (Figure 1A-B). Higher TNF levels were found in the unstimulated supernatant of PMR patients at baseline compared to controls (p=0.0127). Surprisingly, IL-6 levels in the stimulated supernatant of PMR patients at T0 were lower than those of controls (p=0.0426). No significant differences were found in the serum levels of IL-1β and TNF, while IL-6 levels in the sera of PMR patients at baseline were higher compared to those of the control group (p=0.0123).ConclusionPBMCs of PMR patients seem to be activated at baseline compared to controls. Our data indicate a possible role of immunosenescence or an “exhaustion” of PMBCs, that could be constitutively activated and not prone to responding to further stimuli, such as LPS. Moreover, we observed higher serum levels of IL-6 in PMR patients. More studies are needed to confirm the role of PBMCs and inflammatory cytokines in the pathogenesis of PMR.Figure 1.- IL-6 (A) and IL-β (B) levels in the unstimulated supernatant of PMR patients at baseline (PMR T0-) and after 4 months of GC therapy (PMR T4-)Data are shown as Tukey boxplots; lines represent the median level with the 25th-75th percentile.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

BackgroundDietary habits and Physical Activity (PA) are two pivotal features of healthy life in terms of the prevention and treatment of metabolic and chronic disease [1]. Few data are available concerning these issues in isolated Sjӧgren’s syndrome (SS) patients [2].ObjectivesAim of the study was to assess the level of PA, the adherence to Mediterranean diet (MD) and the nutritional status of Italian patients with SS.MethodsConsecutive patients with SS (AECG criteria 2002) were enrolled. For each patient ESSDAI, ESSPRI, SSDDI were calculated. The value of focus score was obtained in the minor salivary gland biopsy (MSG), when performed. The International Physical Activity Questionnaire (IPAQ) was administered to assess the level of physical activity; the Mediterranean Diet (MDiet) score [3] was employed to analyse adherence to Mediterranean diet (the score spans from 0 - no adherence - to 55 - maximum adherence).In a subgroup of 26 patients, the dietary habits were evaluated with a daily food diary. Sarcopenia, a muscle failure that accrue across a lifetime, was identified by a diagnostic algorithm [4] with age-differentiated cut-offs; the Hand Grip test (HGT) and the Chair Stand test (CST) were used as screening tests, the Dual Energy X-ray Absorptiometry (DEXA) was employed to confirm and to quantify Appendicular Skeletal Mass (ASM, kg/m2). The muscular performance (6-minutes Walking Test – 6MWT) was measured to grading sarcopenia.Statistical analysis was performed with SPSS.ResultsThe 130 pSS patients enrolled showed a moderate adherence to the Mediterranean Diet (median MDiet = 33, third quintile) and played a moderately PA (median MET x min/wk in the second tertile). No significant associations were found between BMI, PA, MDiet and clinimetric index of pSS (ESSDAI, ESSPRI, SSDDI).BMI was higher in older patients (R 2 0.96, p 0.02).16 out of the 26 subjects of the subgroup (61%) showed a reduction in muscle strength (HGT and/or CST) and therefore a condition of probable sarcopenia but only 6 (23%) subjects were found to be sarcopenic (ASM). Only one sarcopenic patient had a 6MWT less than 10% of the predicted value and a reduced PA in MET x min/wk.Patients’ daily diet consisted of 43% of total energy coming from carbohydrate, fat represented 40% of total energy intake, the remaining 17% daily energy coming from proteins. Fat consumption is higher and complex carbohydrates intake is lower compared to the levels of energy and nutrient intake for the Italian population [5].ConclusionThis preliminary study considering Italian women with SS shows a moderate adherence to the MD similar to what previously reported in the general female population in Europe (32.5 ± 5 DS) [6].Moreover, considering the results obtained in the subgroup, SS patients seem to have an unbalanced diet because of an intake of fat foods higher than that recommended in the Mediterranean Diet. A possible explanation for the higher fats intake may be a lubricating effect aiding mastication and swallowing. It can be hypothesized that an unbalanced diet might be partly responsible for increasing chronic damage and the associated reduction in muscle mass.References[1]Bonofiglio, Nutrients 2022[2]Ng, Rheumatol Int, 2017[3]Panagiotakos, J Med Food, 2007[4]Cruz-Jentoft, 2019[5]LARN 2014[6]Fallazize, Nutrients 2018Acknowledgements:NIL.Disclosure of InterestsNone Declared.

Background:Digital image analysis (DIA) is increasingly utilized for evaluating salivary gland (SG) histopathology in Sjögren’s disease (SD) [1,2]. DIA, particularly in assessing the area fraction (AF) of lympho-mononuclear cell aggregates, addresses the limitations of the focus score (FS) and minimizes variability between raters and across centers [1]. Utilizing DIA for quantifying intraepithelial B-lymphocytes proves valuable in the assessment of lymphoepithelial lesions [2]. In addition, DIA serves as a precise tool for longitudinally quantification of histopathological parameters before and after treatment, in randomized clinical trials (RCT) [3]. Despite on-going advancements in DIA and its apparent advantages over standard histopathology, its regular implementation is hampered by the absence of standardized protocols for histopathology and DIA parameters.Objectives:The aim is to establish consistency in evaluating salivary gland (SG) histopathology by defining i) standard operating protocols for DIA quantification and ii) identifying histological markers and parameters for monitoring inflammatory changes (Table 1). This harmonization is crucial to implement SG histopathology DIA tools in diagnosing and/or monitoring Sjögren’s disease (SD) histological progression.Methods:Digital slides of Hematoxylin and Eosin (H&E) and immunohistochemistry (IHC) from 21 Sjögren’s disease (SD) patients (11 labial, 10 parotid salivary glands) underwent blind analysis by three independent raters across three centers (UMCG-Groningen, QMUL-London, Rheumatology-Udine). Sequential sections were stained for both H&E and various IHC markers (CD45, CD3, CD20, CD21, CD138, IgG, IgA, and IgM), and the resulting images were shared for analysis. The QuPath open-source software was used to compare assessments for each marker, and the collected data were utilized to calculate the interclass correlation coefficient (ICC) to evaluate the consistency and reproducibility of agreement among the raters (Table 1). Quantitative surrogate markers for T/B cell segregation were evaluated using ROC-curve analysis.Results:A high inter-observer variability was confirmed in the focus score (FS) assessment highlighting a significant improvement when using aggregate area fraction (AF) on H&E staining. The use of CD45 facilitated the quicker identification of focal infiltrates, but did not show a notable enhancement in interclass correlation coefficient (ICC) compared to aggregate AF on H&E. In the quantitative assessment of CD3 (T-cells) and CD20 (B-cells), the percentage of positive cells (of all nucleated cells) exhibited a higher ICC compared to cell-density and positive AF (Table 1). In exploring T/B cell segregation, a key step in the lymphoid organization of inflammatory aggregates in SD, we identified an optimal threshold of 200 B-cells/mm2 to effectively distinguish between tissue sections with segregated and non-segregated aggregates. For identifying aggregates with ectopic lymphoid structure development, the digital count of CD21+ B-cell aggregates demonstrated a higher agreement rate compared to CD21+ AF. Finally, for quantifying plasma cells, the manually determined IgA/IgG shift (≤70% IgA+, ≥30% IgG+) proved to be a more reliable tool than CD138. As a standardization recommendation, we propose quantifying IgG- and IgA-positive plasma cells (as a percentage of positive cells) through digital image analysis (DIA) to objectively calculate the IgA/IgG shift.Conclusion:The proposed workflow for DIA is applicable to both labial and parotid SG, improving the standardization of the quantitative assessment in SG histopathology. This approach identifies histological parameters with high inter-rater agreement across different centers, thereby facilitating the harmonization of SG assessments among diverse cohorts and randomized controlled trials (RCTs).REFERENCES:[1] D. Lucchesi, E. Pontarini, et al. Clin. Exp. Rheumatol. 38 Suppl 1, 180–188 (2020).[2] M. S. van Ginkel, et al. Rheumatology 62, 428–438 (2022).[3] E. Pontarini, et at. Arthritis Rheumatol. (2023), doi:10.1002/art.42772.Acknowledgements:NIL.Disclosure of Interests:Elena Pontarini: None declared, Maria Teresa Rizzo: None declared, Silvia C. Liefers: None declared, Giulia Cavallaro: None declared, Bert van der Vegt: None declared, Serena Colafrancesco: None declared, Alen Zabotti: None declared, Luca Quartuccio: None declared, Alfredo Pulvirenti: None declared, Anwar Tappuni: None declared, Nurhan Sutcliffe: None declared, Salvatore De Vita: None declared, Costantino Pitzalis: None declared, Frans G.M. Kroese: None declared, Michele Bombardieri GSK, Janssen, Janssen, Gwenny M. Verstappen: None declared.

This work presents an analytical approach for studying the cosmological 21cm background signal from the Dark Ages (DA) and subsequent Epoch of Reionization (EoR). We simulate differential observations of a galaxy cluster to demonstrate how these epochs can be studied with a specific form of the Sunyaev-Zel’dovich Effect called the SZE-21cm. This work produces simulated maps of the SZE-21cm and shows that the SZE-21cm can be extracted from future observations with low-frequency radio interferometers such as the Hydrogen Epoch of Reionization Array (HERA) and the Square Kilometre Array (SKA). In order to simulate near realistic scenarios, we look into cosmic variance noise, incorporate and take into account the effects of foregrounds, thermal noise, and angular resolution for our simulated observations. We further extend this exploration by averaging over a sample of galaxy clusters to mitigate the effects of cosmic variance and instrumental noise. The impact of point source contamination is also studied. Lastly, we apply this technique to the results of the EDGES collaboration, which in 2018 reported an absorption feature of the global 21cm background signal centred at 78 MHz. The challenges to be addressed in order to achieve the objectives of this work include errors that arise due to cosmic variation, instrumental noise and point source contamination. Our approach demonstrates the potential of the SZE-21cm as an indirect probe for the DA and EoR, and we conclude that the spectral features of the SZE-21cm from our simulated observations yield results that are close to prior theoretical predictions and that the SZE-21cm can be used to test the validity of the EDGES detection.