Ligation of TLR Homologue CD180 of B Cells Activates the PI3K/Akt/mTOR Pathway in Systemic Sclerosis and Induces a Pathological Shift in the Expression of BAFF Receptors.

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) pathways are known to play a key role in B-cell activation and fibrosis in systemic sclerosis (SSc). Receptors of B-cell activator factor (BAFF) utilize these pathways, which can be influenced by Toll-like receptors (TLRs), as TLRs can alter the expression of BAFF-binding receptors. Our results show that B-cell stimulation via TLR homologue CD180 phosphorylates Akt in diffuse cutaneous SSc (dcSSc) to a lower extent than in healthy controls (HCs). We found basal downregulated BAFF receptor (BAFF-R) and enhanced transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) expression in dcSSc B cells, which might enhance the formation of autoantibody-secreting plasma cells. Moreover, this pathological shift was observed in naive B cells, emphasizing the importance of their increase in SSc. Additionally, we measured higher serum levels of autoantibodies to BAFF in dcSSc patients, suggesting that an imbalance in the complex system of BAFF/anti-BAFF autoantibodies/BAFF-binding receptors may contribute to the development of SSc. Anti-CD180 antibody treatment had opposite effects on the expression of BAFF-R and TACI in HC B cells, resulting in similar levels as observed in SSc B cells without stimulation, which argues against the usefulness of such therapy in SSc.

Sensitivity to change of joint count composite indices in 72 patients with systemic sclerosis.

OBJECTIVES: We validated the responsiveness of joint count composite indices (JCCIs) in 72 patients with systemic sclerosis (SSc). METHODS: Changes in Disease Activity Score of 28 Joints using ESR and CRP (DAS28-ESR, DAS28-CRP), Simplified Disease Activity Index (SDAI) and the Clinical Disease Activity Index (CDAI) were evaluated in a one-year follow-up study. Charts of patients including swollen/tender joint counts, laboratory signs of inflammation, and visual analogue scales referring to disease activity, severity and pain were also blindly categorized by two rheumatologists as improved, unchanged or deteriorated. These categories were used as references for the determination of effect size (ES) and standardised response mean (SRM). RESULTS: Articular inflammation improved in 15, deteriorated in 12, and remained unchanged in 45 (63%) patients with SSc based on the concordant opinion of two clinical investigators. All four JCCIs were sensitive to changes (ES>1; SRM>1). The correlation between changes in JCCIs and the physicians' evaluation was high (r >0.68; p<0.001). Arthritis was predominantly prone to change in patients with high JCCIs, impaired functional status, anti-RNA polymerase III antibodies and patients on DMARD therapy. Synovitis was more prevalent in patients with early diffuse SSc, and tended to improve during the follow-up. CONCLUSIONS: All four JCCIs were sensitive to changes, if tender/swollen joints were present at baseline. Articular inflammation was most prone to change in patients with high JCCIs, impaired functional status and already decreased health-related quality of life at baseline.

Analysis of PI3K Pathway Associated Molecules Reveals Dysregulated Innate and Adaptive Functions of B Cells in Early Diffuse Cutaneous Systemic Sclerosis.

B cell activation is an early event in the development of systemic sclerosis (SSc). The classical activation of B cells downstream of the B-cell receptor (BCR) involves the phosphatidylinositol-3 kinase (PI3K) pathway that integrates the effects of multiple co-stimulatory receptors. Our analysis of PI3K pathway associated molecules in peripheral blood B cells of early diffuse cutaneous SSc (dcSSc) patients showed altered mRNA expression of Toll-like receptor (TLR) homolog CD180, TLR4, complement component 3, IL-4 receptor and secreted phosphoprotein 1 (SPP1). Parallel to this, we found elevated basal SPP1 secretion in dcSSc B cells, but, with BCR + IL-4 receptor co-stimulation, we could not induce further secretion. CD180 stimulation alone resulted in NF-κB activation in more B cells than CD180 + BCR co-stimulation both in dcSSc and healthy control (HC), but the co-engagement increased the phosphorylation of NF-κB only in dcSSc B cells. Additionally, in contrast with HC B cells, the lower basal production of IL-10 by dcSSc B cells could not be elevated with CD180 stimulation. Furthermore, activation via CD180 increased the percentage of CD86+ switched memory (CD27+IgD-) B cells in dcSSc compared to HC. Our results suggest that alternative B cell activation and CD180 dysfunction cause imbalance of regulatory mechanisms in dcSSc B cells.

Toll-Like Receptor Mediated Activation of Natural Autoantibody Producing B Cell Subpopulations in an Autoimmune Disease Model.

Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180+ cells was the highest in the CD19+CD27+IgD+ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.

Impairment of Left Atrial Mechanics Is an Early Sign of Myocardial Involvement in Systemic Sclerosis.

BACKGROUND: Left ventricular (LV) diastolic dysfunction is common in systemic sclerosis (SSc). Less is known, however, about left atrial (LA) mechanics in this context. The aim of this study was to investigate the correlation between LV diastolic function and LA mechanics in SSc patients with the use of volumetric and 2-dimensional speckle tracking-derived strain techniques and to compare the results with those obtained in healthy subjects. METHODS AND RESULTS: Seventy-two SSc patients and 30 healthy volunteers (H) were investigated. LV diastolic function was classified as normal (I), impaired relaxation (II), and pseudonormal pattern (III). LA reservoir (H: 51.8 ± 7.4%; I: 45.1 ± 8.1%; II: 42.2 ± 6.6%; III: 36.6 ± 7.3%; analysis of variance: P < .001) and contractile strain (H: 24.8 ± 4.9%; I: 18.2 ± 4.4%; II: 21.5 ± 2.8%; III: 16.8 ± 3.6%; P < .001) already showed significant worsening in SSc patients with preserved LV diastolic function compared with healthy subjects. LA conduit strain (H: 27.1 ± 4.6%; I: 26.9 ± 5.7%; II: 20.6 ± 6.1%; III: 19.5 ± 5.3%; P < .001) was preserved in this early phase. Further deterioration of reservoir strain was pronounced in the pseudonormal group only. LA contractile strain increased significantly in the impaired relaxation group and then decreased with the further worsening of the LV diastolic function. Regarding phasic volume indices, the differences between groups were not always statistically significant. CONCLUSION: LA mechanics strongly reflects the changes in LV diastolic function in SSc. On the other hand, strain parameters of the LA reservoir and contractile function already show significant worsening in SSc patients with preserved LV diastolic function, suggesting that impairment of the LA mechanics is an early sign of myocardial involvement in SSc.

The European Scleroderma Trials and Research group (EUSTAR) task force for the development of revised activity criteria for systemic sclerosis: derivation and validation of a preliminarily revised EUSTAR activity index.

BACKGROUND: Validity of European Scleroderma Study Group (EScSG) activity indexes currently used to assess disease activity in systemic sclerosis (SSc) has been criticised. METHODS: Three investigators assigned an activity score on a 0-10 scale for 97 clinical charts. The median score served as gold standard. Two other investigators labelled the disease as inactive/moderately active or active/very active. Univariate-multivariate linear regression analyses were used to define variables predicting the 'gold standard', their weight and derive an activity index. The cut-off point of the index best separating active/very active from inactive/moderately active disease was identified by a receiver-operating curve analysis. The index was validated on a second set of 60 charts assessed by three different investigators on a 0-10 scale and defined as inactive/moderately active or active/very active by other two investigators. One hundred and twenty-three were investigated for changes over time in the index and their relationships with those in the summed Medsger severity score (MSS). RESULTS: A weighted 10-point activity index was identified and validated: Δ-skin=1.5 (Δ=patient assessed worsening during the previous month), modified Rodnan skin score (mRss) >18=1.5, digital ulcers=1.5, tendon friction rubs=2.25, C-reactive protein >1 mg/dL=2.25 and diffusing capacity of the lung for CO (DLCO) % predicted <70%=1.0. A cut-off ≥2.5 was found to identify patients with active disease. Changes in the index paralleled those of MSS (p=0.0001). CONCLUSIONS: A preliminarily revised SSc activity index has been developed and validated, providing a valuable tool for clinical practice and observational studies.

The presence of small joint contractures is a risk factor for survival in 439 patients with systemic sclerosis.

OBJECTIVES: Analysis of risk factors and mortality of 439 patients with systemic sclerosis (SSc) in a tertiary care centre. METHODS: The mean follow up time was 8.4±5.6 years. Lost to follow up rate was 6.4%. Female to male ratio was 366 to 73. Two hundred sixty patients had limited and 179 diffuse cutaneous SSc (dcSSc). A standard protocol including musculoskeletal examinations was used for the assessment of patients. RESULTS: By Kaplan-Meier analysis the overall 5-, 10- and 15 year survival were 88.2%, 79.9% and 73.6%, respectively. Univariate analysis showed that dcSSc, male gender, presence of small joint contractures, pulmonary interstitial, cardiac, oesophageal involvement, scleroderma renal crisis, arterial hypertension, anti-topoisomerase antibody, anemia, hypalbuminemia, coexistent malignancies and elevated erythrocyte sedimentation were associated with poor survival. Lack of giant capillaries, avascular zones or neo-angiogenesis on capillaroscopy, and presence of anti-centromere antibodies were associated with favourable outcome. Multivariate regression analysis showed presence of small joint contractures, history of arterial hypertension, male gender, diffusing capacity of carbon monoxide <50%, right ventricular pressure >40 mmHg on echocardiography, less than 50% ejection fraction, anti-topoisomerase I positivity, anemia, and serum albumin concentration < 35 g/l as well as current or history of coexistent malignancy were independent poor prognostic factors. CONCLUSIONS: In addition to well-known factors predicting poor outcome in SSc, the presence of small joint contractures was a newly identi ed independent risk factor of mortality. Our data also confirmed a recent finding showing that history of arterial hypertension was also a poor prognostic factor.

Efficacy of intensive hand physical therapy in patients with systemic sclerosis.

OBJECTIVES: To evaluate the efficacy of a three-week period of complex and intensive hand physical therapy on hand function in patients with systemic sclerosis (SSc). METHODS: Thirty-one patients with SSc were treated. Hand stretching exercises, ergotherapy supplemented with thermal and mud baths, whirlpool therapy and soft tissue massage were daily used during a three-week period. The control SSc group (n=22) received similar management for their large joints leaving out their hands. The primary outcomes of this study were the Health Assessment Questionnaire (HAQ) and the Disabilities of the Arm, Shoulder and Hand (DASH). Hand Anatomic Index (HAI), Cochin Hand Function (CHFT) and clinical characteristics were also assessed before starting the therapy and six months afterwards. RESULTS: Six months after the investigation period, only the group receiving hand physical therapy showed improvement in HAQ and DASH scores compared to the baseline values (p<0.05). The improvement in median HAQ value (25%-75% quartiles) reached the clinical meaningful rate (baseline 1.125/0.625-1.625/ versus 0.75/0.25-1.5/ at six months). Visual analogue scales of global pain (p<0.01) and Raynaud's phenomenon (p<0.05) also had better results than at baseline. HAI, gripping strength and CHFT also showed some improvement, but did not reach the significance level of change by the end of the six-month observation period. CONCLUSIONS: The complex physical therapy caused favourable changes in both the HAQ and the DASH indicating that this particular program had some long-term beneficial effect on hand function in patients with SSc.

Validation of disease activity indices using the 28 joint counts in systemic sclerosis.

OBJECTIVES: To validate the Disease Activity Score 28 using ESR (DAS28-ESR) and CRP (DAS28-CRP), the Simplified Disease Activity Index and the Clinical Disease Activity Index used in RA for SSc patients. METHODS: Seventy-seven SSc patients, 40 RA patients, 20 patients with primary RP (PRP) and 28 healthy volunteers were assessed. Besides the disease activity composite indices, the European Scleroderma Study Group Activity Index (EScSG-AI), the HAQ-DI, the Cochin Hand Function Scale and the Short Form Health Survey (SF36) were evaluated. The validation procedure included the assessment for truth, discrimination and feasibility. RESULTS: DAS28-ESR, DAS28-CRP, Simplified Disease Activity Index and Clinical Disease Activity Index showed significant correlation with EScSG-AI, HAQ-DI, Cochin Hand Function Scale and the physical component of SF36 (P < 0.001). All four indices discriminated patients with SSc from RA, PRS and healthy controls, respectively (P < 0.01). With the exception of DAS28-CRP, the other three indices also discriminated between subgroups of SSc based on value of EScSG-AI (⩽3 and >3) (P < 0.05). All four disease activity composite indices showed a good inter- and intraobserver reliability based on repeated measures of two independent investigators (P < 0.001). CONCLUSION: All four disease activity composite indices were found to be valid measures for assessing arthritis in SSc. DAS28-ESR showed the best performance regarding reliability and construct validity.

Reduced non-switched memory B cell subsets cause imbalance in B cell repertoire in systemic sclerosis.

OBJECTIVES: Analysis of peripheral blood B lymphocytes in patients with systemic sclerosis (SSc) has provided evidence for specific alterations in naive and memory B cell balance. However, memory B cell subsets in SSc have not been thoroughly investigated. This study sought to identify phenotypic abnormalities and activation markers in peripheral blood memory B cells in SSc subtypes. METHODS: Blood samples were obtained from 28 SSc patients with early form of disease (9 limited (lcSSc), 19 diffuse cutaneous SSc (dcSSc)) and 15 healthy controls. After magnetic bead separation of CD19+ B cells, multiparametric flow cytometry was performed and CD19+CD27- IgD+ naive, CD19+CD27+ memory, CD19+CD27+IgD+ non-switched memory CD19+CD27+IgD- switched memory, CD19+CD27-IgD- double negative (DN) memory, CD80+ or CD95+ activated cells were identified. RESULTS: The proportion of naive B cells was higher (p=0.046) in SSc than in controls, with a decreased percentage of memory (p=0.018), especially non-switched memory B cells (p=0.015). The dcSSc patients had a significantly higher frequency of switched memory and DN memory B cells compared to lcSSc patients (p=0.025 and p=0.031). Percentage of CD95+CD27+ memory and CD95+ DN memory B cells was also significantly elevated in dcSSc compared to lcSSc patients (p=0.038 and p=0.045). CONCLUSIONS: We conclude that the decreased proportion of memory B cells in SSc is due to reduction of non- switched memory B cells, resulting in an imbalance between the tolerogenic and activated memory B cell types. Elevated switched and activated CD95+ DN memory B cells may serve as a biomarker for dcSSc and can have a pathogenic potential by cytokine and autoantibody production.

Preliminary analysis of the very early diagnosis of systemic sclerosis (VEDOSS) EUSTAR multicentre study: evidence for puffy fingers as a pivotal sign for suspicion of systemic sclerosis.

OBJECTIVES: The EULAR (European League Against Rheumatism) Scleroderma Trials and Research Group (EUSTAR) has identified preliminary criteria for very early diagnosis of systemic sclerosis (SSc). Our aim was to assess the prevalence of each proposed diagnostic item in a large observational patient cohort with Raynaud's phenomenon (RP). METHODS: Baseline data of 469 RP patients enrolled into the Very Early Diagnosis of Systemic Sclerosis (VEDOSS) cohort are presented. RESULTS: 68% of all RP patients were antinuclear antibody (ANA) positive. ANA+ RP patients more frequently had previous or current puffy fingers (PuFi) (38.5% and 23.3%, p<0.01) and an SSc pattern on nailfold capillaroscopy (NC) (53.6% and 13.4%, p<0.001) than ANA- patients. Telangiectasia, current digital ulcers and digital pitting scars were also commoner in ANA+ RP patients. 38% of ANA+ patients presented with all three features, which should raise suspicion of very early SSc (ANA+RP+PuFi constitutes a 'red flag'). These patients more frequently exhibited an NC SSc pattern, sclerodactyly and telangiectases compared to ANA+ patients without PuFi. Almost 90% of patients with 'red flags' had anti-centromere or anti-topoisomerase I antibodies and/or an NC SSc pattern, and fulfilled the EUSTAR criteria for very early SSc. Previous or current PuFi were present in 23.3% of ANA- RP patients, eight of whom also had an NC SSc pattern. CONCLUSIONS: In addition to well-characterised predictive factors, PuFi is an important sign raising suspicion for underlying very early SSc in patients with RP. The relevance of PuFi in ANA- RP patients should be clarified.

Clinical usefulness of measuring red blood cell distribution width in patients with systemic sclerosis.

OBJECTIVE: Red blood cell distribution width (RDW) is a biomarker quantifying the variability of red blood cell size in peripheral blood. Elevated RDW has been found to be an independent prognostic factor for cardiovascular events. SSc is characterized by generalized micro- and macroangiopathy. Our aim was to investigate RDW as a potential biomarker for the assessment of the severity of vascular involvement. METHODS: One hundred and sixty-eight consecutive SSc patients--62 with dcSSc and 106 with lcSSc--were investigated at baseline and after 1-year of follow-up. Measurements in 93 patients with primary RP and 40 healthy subjects served as controls. RESULTS: The median RDW value of patients with SSc was higher [14.2% (25th-75th percentiles 13.5-14.8%) compared with the group of primary RP patients [13.9% (13.4-14.4%); P < 0.05) and healthy volunteers [13.6% (13.2-13.8%; P < 0.01]. dcSSc and anti-topoisomerase antibody-positive cases showed elevated RDW values compared with lcSSc and anti-topoisomerase antibody-negative cases (P < 0.05). RDW showed a positive correlation with inflammatory markers, including ESR (P < 0.05) and CRP (P < 0.05), and a negative correlation with forced vital capacity (P < 0.05) and diffusing capacity of the lung for carbon monoxide (DLCO) (P < 0.05) during the follow-up. An increase in RDW of >5% during follow-up was associated with an average 8.9% decrease in left ventricular ejection fraction (LVEF) and 7% in DLCO and these associations were independent of each other. CONCLUSION: RDW in SSc may represent an integrative measure of multiple pathological processes including extensive vasculopathy, fibrosis or ongoing inflammation. An increase in RDW may indicate an impairment of cardiorespiratory function.

Characteristics of ScleroID highlighting musculoskeletal and internal organ implications in patients afflicted with systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a multi-organ disease with impaired health-related quality of life (HRQoL). The EULAR SSc Impact of Disease (ScleroID) is a newly introduced SSc-specific patient-reported outcome to evaluate HRQoL in SSc. OBJECTIVE: To investigate the correlation between the ScleroID and the involvement of organ systems as well as disease activity/damage in a SSc cohort from a large tertiary care centre. PATIENTS AND METHODS: The ScleroID and clinical characteristics including internal organ involvement and hand function were investigated in 160 consecutive patients with SSc (median age 46 (43;56) years; diffuse cutaneous SSc 55%). RESULTS: A strong correlation was found between the ScleroID and articular disease activity scores (DAS28-CRP, DAS28-ESR, CDAI, SDAI), a hand function performance test, the Hand Anatomy Index and muscle strength tests. Additionally, a strong significant correlation was discovered using instruments representing hand function and musculoskeletal disability including the Cochin Hand Function Scale, the Quick Questionnaire of the Disability of the Hands, Arms and the Shoulders and the Health Assessment Questionnaire Disability Index. A significant negative correlation was found between the ScleroID score and the 6-min walking test (6MWT) (rho - 0.444, p < 0.001). Clinically mild lung/heart disease did not show increased ScleroID values. The Mouth Handicap in the Scleroderma Scale and the University of California Los Angeles Scleroderma Clinical Trials Consortium gastrointestinal tract 2.0 also showed significant positive correlations to the ScleroID score (rho: 0.626, p < 0.001; rho: 0.646, p < 0.001, respectively). Patients experiencing oesophageal difficulties bore a significantly higher score compared to individuals with a normal functioning oesophagus (3.2/1.5;4.5/ vs. 2.2/1.0;3.2/, p = 0.011). Moreover, the ScleroID showed a significant positive correlation to the revised EUSTAR disease activity index and modified activity index. CONCLUSION: In a large single-centre cohort, the previously described ScleroID-related findings were confirmed. Furthermore, several organ involvement-related functional and performance tests showed a good correlation to the ScleroID including the 6MWT and gastrointestinal-related complaints. Many aspects of musculoskeletal damage, overall disease activity, pain and fatigue were also well represented in the ScleroID, which efficiently reflects the impact of organ involvement, disease activity and functional damage.

Minimal Clinically Important Differences (MCID) for the Functional Assessment of Chronic Illness Therapy Fatigue Scale in Patients with Systemic Sclerosis.

(1) Background: Systemic sclerosis (SSc) is characterized by significant fatigue, causing diminished quality of life (QoL). The aim of this study was to examine fatigue levels and their associations with clinical factors and determine the minimal clinically important difference (MCID) value for the Functional Assessment of Chronic Illness Therapy Fatigue Scale (FACIT-FS). (2) Methods: A total of 160 SSc patients and 62 individuals without SSc were followed-up over a 12-month period by measuring the FACIT-FS and the Visual Analogue Scale and the Short Form 36 Vitality Score analyzing changes in exhaustion. (3) Results: Fatigue was strongly correlated with HRQoL, level of pain, emotional disorders, physical capability and functionality. The MCID values for FACIT-FS were calculated as -3 for deterioration and +4 for improvement after a 12-month follow-up. The predictors of improvement of fatigue from baseline parameters were the significant disease activity, the patients' poorer functionality and the short disease duration. Patients with scleroderma-related interstitial lung disease at baseline had approximately tripled risks for worsening fatigue. The independent influential factors regarding the changing of FACIT-FS were improving or worsening in the same direction in reference to physical condition, gastrointestinal and emotional factors. (4) Conclusions: Fatigue is a multi-dimensional symptom, which is strongly correlated to HRQoL. MCID values of FACIT-FS can be useful tools in monitoring the changes of HRQoL in clinical trials and in daily practice among patients with SSc.

Mechanism of coronary flow reserve reduction in systemic sclerosis: insight from intracoronary pressure wire studies.

OBJECTIVE: Functional impairment of coronary microcirculation is thought to be a major pathway in the development of primary cardiac involvement in SSc; however, the underlying mechanism is not fully understood. We aimed to investigate the mechanisms of coronary flow reserve (CFR) reduction in patients with SSc. METHODS: Seventeen SSc patients and 17 gender- and age-matched controls were enrolled. Coronary angiography and determination of coronary flow parameters including index of myocardial resistance (IMR) using intracoronary pressure wire at basal conditions and during vasodilator-induced maximal hyperaemia were performed. Transit times of repeated intracoronary saline injection were measured to evaluate the role of cold exposure. RESULTS: SSc patients with decreased CFR had accelerated basal coronary flow velocity (P < 0.05), and their IMR in hyperaemia (IMR(hyp)) did not differ from either SSc patients with normal CFR or from the controls (P = 0.292 and P = 0.308). The coronary flow velocity of SSc patients correlated with the IMR at baseline (IMR(bas)) (r = 0.56, P = 0.019). Injection of room temperature saline did not provoke changes in coronary transit times. CONCLUSIONS: The lack of decrease in the maximal vasodilatation response indicates that there is no irreversible functional damage at the level of the coronary arterioles. In patients with reduced CFR, the decreased basal IMR and higher velocity reflect compensatory vasodilatory mechanisms probably triggered by ischaemic signals deriving from abnormal myocardial microcirculation.

Increased Frequency of Activated Switched Memory B Cells and Its Association With the Presence of Pulmonary Fibrosis in Diffuse Cutaneous Systemic Sclerosis Patients.

Disease-associated, high-affinity pathological autoantibody production is a well-described consequence of immune dysregulation affecting B cells in systemic sclerosis (SSc), including the distribution of B-cell subsets. We have previously shown that the increased relative frequency of CD19+CD27+IgD- switched memory B cells is associated with the severe form of SSc. This study sought to analyze memory B cell subsets using an extended range of markers for further subdivision based on CD19, IgD, CD27, CD38 and CD95 phenotype, to define relationship between the alterations of memory B cell subsets and the clinical features of SSc. Peripheral blood samples were obtained from 21 SSc patients, including 14 diffuse (dcSSc) and 7 limited (lcSSc) cutaneous SSc patients, with disease duration of 2.7 ( ± 1.6) years. After purification of CD19+ B cells, multiparametric flow cytometry was performed and the frequencies of CD19+IgD-CD27-CD38+ double negative (DN) 1, CD19+IgDloCD27+CD38+ unswitched, CD19+IgD-CD27+CD38+CD95- resting switched and CD19+IgD-CD27+CD38-CD95+ activated switched memory (ASM) B cells were determined, and correlated with clinical features of SSc. The dcSSc patients had a higher frequency of ASM B cells (p = 0.028) compared to lcSSc patients. The percentage of ASM B cells was elevated in anti-Scl-70 (anti-topoisomerase I) antibody positive patients compared to negative patients (p = 0.016). Additionally, the frequency of ASM B cells was also increased in patients with pulmonary fibrosis (p = 0.003) suggesting that patients with severe form of SSc have higher ASM B cell ratios. Furthermore, the ratio of DN1 B cells was decreased (p = 0.029), while the level of anti-citrate synthase IgG natural autoantibody was elevated (p = 0.028) in patients with active disease. Our observations on the increase of ASM B cells in dcSSc and in patients with pulmonary fibrosis may point to the association of this alteration with the severe form of the disease. Functionally the correlation of ASM B cells as effector memory-plasma cell precursors with anti-topoisomerase I antibody positivity could reflect their contribution to pathological autoantibody production, whereas the decrease of memory precursor DN B cells and the increase of anti-citrate synthase IgG autoantibody may have potential significance in the assessment of disease activity.

Progression of patients with Raynaud's phenomenon to systemic sclerosis: a five-year analysis of the European Scleroderma Trial and Research group multicentre, longitudinal registry study for Very Early Diagnosis of Systemic Sclerosis (VEDOSS).

BACKGROUND: Preliminary criteria for the very early diagnosis of systemic sclerosis (VEDOSS) have been previously proposed to identify signs and symptoms in patients with Raynaud's phenomenon. Patients with all signs or symptoms of the VEDOSS criteria already fulfil the 2013 American College of Rheumatology-European League Against Rheumatism (ACR-EULAR) classification criteria for systemic sclerosis. However, prospective data for the evolution to fulfilling these criteria do not exist. We therefore aimed to determine the clinical value of the VEDOSS criteria to identify patients with Raynaud's phenomenon who progress to systemic sclerosis within 5 years. METHODS: The VEDOSS project was a multicentre, longitudinal registry study done in 42 European Scleroderma Trial and Research group centres located in 20 countries in Europe, North America, and South America. Patients with Raynaud's phenomenon were eligible for enrolment. Those who had fulfilled the 1980 ACR or the 2013 ACR-EULAR classification criteria for systemic sclerosis, as well as of any other ACR or EULAR classification criteria for other definite connective tissue diseases at enrolment were excluded. Data were recorded each year during follow-up visits and included the four VEDOSS criteria (ie, positivity for antinuclear antibodies [ANAs], puffy fingers, systemic sclerosis-specific autoantibodies, and abnormal nailfold capillaroscopy). The primary endpoint was the fulfilment of the 2013 ACR-EULAR classification criteria for systemic sclerosis (ie, progression from enrolment to follow-up). Proportion of progressors and VEDOSS criteria interaction were reported descriptively. Predictors of progression of the distinct VEDOSS criteria interactions were determined based on the point prevalence at 5 years. To investigate the intermediate course of progression of the distinct VEDOSS criteria and their combinations, Kaplan-Meier analysis was done. RESULTS: Between March 1, 2010, and Oct 4, 2018, we enrolled 1150 patients with Raynaud's phenomenon in the VEDOSS database. 764 (66·4%) of 1150 patients met the VEDOSS criteria for study inclusion. Of the 764 patients, 553 (72·4%) had at least one available follow-up visit and the median duration of follow-up was 3·6 years (IQR 1·7-5·8). The mean age was 45·9 years (SD 15·0), 507 (91·7%) of 553 participants were female, and the median time since the onset of Raynaud's phenomenon was 4·0 years (IQR 1·7-10·0). At baseline, 401 (73·7%) of 544 patients with Raynaud's phenomenon had detectable ANA, with 208 (39·5%) of 527 patients positive for systemic sclerosis-specific autoantibodies. Nailfold capillaroscopy abnormalities were present in 182 (36·0%) of 505 patients and puffy fingers were detected in 96 (17·8%) of 540 at baseline. 1885 follow-up visits were recorded. 254 (45·9%) of 553 patients completed the study with progression or a 5-year follow-up; of whom, 133 reached the primary endpoint, resulting in an overall progression rate of 52·4%. The absence of ANA at baseline was the factor most strongly associated with a lack of progression within 5 years, with only four (10·8%) of 37 ANA-negative patients progressing. Conversely, positivity at baseline for systemic sclerosis-specific autoantibodies and puffy fingers was the combination having the highest risk of progression (16 [94·1%] of 17). INTERPRETATION: Our results from the VEDOSS project offers a useful tool for a stratified risk approach to patients with Raynaud's phenomenon. The absence of ANA is a strong protective factor that identifies patients with very low risk of developing systemic sclerosis whereas the presence of one or two VEDOSS criteria in patients with Raynaud's phenomenon confers a progressively higher risk for systemic sclerosis over time. This stratification tool can be used both for clinical management and to inform early interventional trials. FUNDING: European Scleroderma Trial And Research and World Scleroderma Foundation.

Cost-of-illness of patients with systemic sclerosis in a tertiary care centre.

OBJECTIVE: The aim of our study was to assess the costs of SSc and to analyse cost drivers. METHODS: A cross-sectional survey of consecutive patients with SSc was performed in a rheumatology centre in Hungary. Clinical characteristics, the European Scleroderma Study Group activity index, disease severity scale (DSS), scleroderma HAQ (S-HAQ) and health care utilization were recorded. Cost calculation was performed and correlation with clinical variables was analysed. Results were compared with RA and PsA. RESULTS: Eighty patients were involved: 72 (90%) women, mean age (s.d.) 57.4 (9.6) years and disease duration of 6.2 (6.6) years and 25% of the cases had dcSSc. Mean total cost was 9619 (s.d. 6444) euros/patient/year with rate of indirect cost being 56%. Disability-related productivity loss (55.2%) and hospitalization (28.3%) were the highest among the cost items. Patients with dcSSc had significantly higher direct costs (P = 0.005) compared with the lcSSc subset. Disease activity showed significant correlation with total costs, DSS and S-HAQ with direct costs. SSc-related costs were higher than in matched RA and PsA cases. CONCLUSIONS: The cost-of-illness of SSc is high with a dominance of productivity loss related costs. Moreover, the disease activity is an important cost driver.

Construct validity evaluation of the European Scleroderma Study Group activity index, and investigation of possible new disease activity markers in systemic sclerosis.

OBJECTIVES: To evaluate the construct validity of the European Scleroderma Study Group (EScSG) activity index and to propose modifications if necessary. METHODS: One hundred and thirty-one consecutive patients were investigated and re-evaluated 1 year later. Modified Rodnan skin score (MRSS), skin ulcers and joint contracture numbers, hand anatomic index (HAI), BMI, spirometry, carbon monoxide diffusing capacity (DL(CO)), left ventricular ejection fraction, pulmonary arterial hypertension, HAQ Disability Index (HAQ-DI), patient skin self-assessment questionnaire and several biomarkers were recorded, in addition to the data required for the EScSG activity index. Statistical analysis was performed by categorical principal component analysis (CATPCA). RESULTS: The EScSG activity index appeared in the same dimension as the HAQ-DI, ulcer score and joint contractures, MRSS, patient-reported skin score and HAI by CATPCA. Parameters of lung involvement appeared in another dimension. We constructed a 12-point activity index that was equally associated with both dimensions, by adding the forced vital capacity/DL(CO), change in DL(CO), change in the ulcer scores, HAQ-DI and patient-reported skin score. Biomarkers including vascular endothelial growth factor, soluble P-selectin glycoprotein ligand-1, CRP and albumin were related to both the EScSG and the 12-point index, though they did not improve the total variance of the model. CONCLUSION: The construct validity of the EScSG activity index is good, though the lung-related disease activity may not be sufficiently represented. Further validation steps may be required for both the EScSG and our 12-point activity index.

Naturally occurring and disease-associated auto-antibodies against topoisomerase I: a fine epitope mapping study in systemic sclerosis and systemic lupus erythematosus.

Auto-antibodies against topoisomerase I (topo I) are frequently detected in sera of systemic sclerosis (SSc) patients. Anti-topo I auto-antibodies are considered to be associated with the diffuse cutaneous form of systemic sclerosis (dcSSc). However, anti-topo I auto-antibodies are also detected in limited cutaneous systemic sclerosis (lcSSc) and systemic lupus erythematosus (SLE). In this study, we compared the epitope specificity of anti-topo I auto-antibodies present in sera of dcSSc, lcSSc and SLE patients. We have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with IgG purified from patients' sera. Regions of topo I selected from the library were expressed as recombinant fusion proteins and were tested with ELISA and western blot. We unexpectedly found that antibodies against a fragment of topo I [fragment F4 [amino acid (AA)] 451-593] could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than SSc and SLE. Using sera of dcSSc, lcSSc and SLE patients, we showed that the pattern of recognized epitopes is different between these patient groups. Fragment F4 was recognized by all patients. Fragment F1 (AA 5-30) was recognized by 9 of 34 dcSSc patients. Fragment F8 (AA 350-400) was recognized by four of eight SLE patients. Analysis of clinical data revealed a significant difference between the F1-negative and F1-positive groups of SSc patients in age and in the duration of the disease. According to our results, the newly identified fragments F1 and F8 could represent characteristic epitopes for dcSSc and SLE, respectively.