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Abstract Background Anti-Jo-1 autoantibodies represent essential markers in the diagnosis of antisynthetase syndrome (ASS). In this retrospective study, we aimed to investigate whether their concentrations and fluctuations could both respectively reflect the severity and evolution of ASS. Methods Between 2015 and 2020, clinical and biological features of ASS patients with at least one positive measure of anti-Jo-1 autoantibody were collected. At each serum sampling, we assessed myositis activity by using the Myositis Intention to Treat Activities Index (MITAX) and compared anti-Jo-1 concentrations with ASS severity, anti-Jo-1 concentrations between patients with and without active disease, and changes in anti-Jo-1 concentrations with disease activity. Results Forty-eight patients with ASS had at least one positive determination of anti-Jo-1 concentration. Among them, twenty-nine patients had at least two determinations of anti-Jo-1 autoantibody in their follow-up. We showed that these autoantibody concentrations were significantly correlated with MITAX (r = 0.4, p = 0.03) and creatine kinase concentration (r = 0.34, p = 0.002) and that they were significantly higher in patients with active disease than in those with inactive disease (91.7 IU/L vs 44.4 IU/L, p = 0.016). During follow-up, we found a significant correlation between fluctuations of anti-Jo-1 autoantibody concentrations and MITAX score (r = 0.7, p

Background: The positivity of anti-RNP autoantibodies as biological criteria for the diagnosis of mixed connective tissue disease (MCTD) has recently divided the rheumatology community. Autoantigenicity of the U1-snRNP complex tends to generate multiple autoantibodies against RNP-A, -C and -70 KDa or Sm proteins. The aim of this study is to identify the most informative autoantibodies in clinical practice, in particular, to contribute to differential diagnosis between MCTD and systemic lupus erythematosus (SLE). Methods: Sera from 74 patients positive for anti-RNP autoantibodies were selected over a period of one year of laboratory practice. Autoantibodies directed against extractable nuclear antigen, RNP proteins (A, C, 70 KDa) and 40 kDa fragments of RNP-70 KDa were investigated by using quantitative fluoroenzymatic assay and Western blot analysis. Results: Among the 74 patients, 40 patients were diagnosed with SLE, 20 with MCTD, six with another autoimmune disease, three with SARS-CoV-2 infection, three with cancer and two were healthy. No preferential clinical association of IgG or IgM autoantibodies directed against each of the RNP proteins was found between SLE and MCTD. In contrast, the proportion of autoantibodies directed against the RNP component within the U1-snRNP complex showed a significantly higher RNP index in patients with MCTD than in those with SLE (p = 0.011), with good performance (sensitivity: 69.2%, specificity: 88.9%). Conclusions: The analysis of the proportion of the different autoantibodies directed against the U1-snRNP complex is more informative than the analysis of each autoantibody separately. A follow-up of patients could be informative about the interest of the RNP index as a predictor of disease evolution.

Abstract Rationale Glioblastoma multiforme (GBM) is a primary brain tumor with poor prognosis. The U.S. food and drug administration approved the use of the anti-VEGF antibody bevacizumab in recurrent GBM. However, resistance to this treatment is frequent and fails to enhance the overall survival of patients. In this study, we aimed to identify novel mechanism(s) responsible for bevacizumab-resistance in CD146-positive glioblastoma. Methods The study was performed using sera from GBM patients and human GBM cell lines in culture or xenografted in nude mice. Results We found that an increase in sCD146 concentration in sera of GBM patients after the first cycle of bevacizumab treatment was significantly associated with poor progression free survival and shorter overall survival. Accordingly, in vitro treatment of CD146-positive glioblastoma cells with bevacizumab led to a high sCD146 secretion, inducing cell invasion. These effects were mediated through integrin αvβ3 and were blocked by mucizumab, a novel humanized anti-sCD146 antibody. In vivo, the combination of bevacizumab with mucizumab impeded CD146 + glioblastoma growth and reduced tumor cell dissemination to an extent significantly higher than that observed with bevacizumab alone. Conclusion We propose sCD146 to be 1/ an early biomarker to predict and 2/ a potential target to prevent bevacizumab resistance in patients with glioblastoma.

Abstract Whereas the detection of antiphospholipid autoantibodies (aPL) in COVID-19 is of increasing interest, their role is still unclear. We analyzed a large aPL panel in 157 patients with COVID-19 according to the disease severity. We also investigated a potential association between aPL and extracellular DNA (exDNA, n = 85) or circulating markers of neutrophil extracellular traps (NET) such as citrullinated histones H3 (CitH3, n = 49). A total of 157 sera of patients infected by SARS-CoV-2 were collected. A large aPL panel including lupus anticoagulant, anti-cardiolipin and anti-beta-2 glycoprotein I (IgG, IgM and IgA), anti-phosphatidylethanolamine IgA, anti-prothrombin (IgG and IgM) was retrospectively analyzed according to the disease severity. We found a total aPL prevalence of 54.8% with almost half of the cases having aCL IgG. Within an extended panel of aPL, only aCL IgG were associated with COVID-19 severity. Additionally, severe patients displayed higher CitH3 levels than mild patients. Interestingly, we highlighted a significant association between the levels of aCL IgG and exDNA only in aCL positive patients with severe disease. In conclusion, we showed a significant link between aPL, namely aCL IgG, and circulating exDNA in patients with severe form of COVID-19, that could exacerbate the thrombo-inflammatory state related to disease severity.

Abstract Background Hypocomplementemic urticarial vasculitis (HUV) is a rare systemic vasculitis. We aimed to describe the kidney involvement of HUV in a multicenter national cohort with an extended follow-up. Methods All patients with HUV (international Schwartz criteria) with a biopsy-proven kidney involvement, identified through a survey of the French Vasculitis Study Group (FVSG), were included. A systematic literature review on kidney involvement of HUV was performed. Results Twelve patients were included, among whom 8 had positive anti-C1q antibodies. All presented with proteinuria, from mild to nephrotic, and 8 displayed acute kidney injury (AKI), requiring temporary haemodialysis in 2. Kidney biopsy showed membrano-proliferative glomerulonephritis (MPGN) in 8 patients, pauci-immune crescentic GN or necrotizing vasculitis in 3 patients (with a mild to severe interstitial inflammation), and an isolated interstitial nephritis in 1 patient. C1q deposits were observed in the glomeruli (n = 6), tubules (n = 4) or renal arterioles (n = 3) of 8 patients. All patients received corticosteroids, and 9 were also treated with immunosuppressants or apheresis. After a mean follow-up of 8.9 years, 6 patients had a preserved renal function, but 2 patients had developed stage 3–4 chronic kidney disease (CKD) and 4 patients had reached end-stage kidney disease (ESKD), among whom 1 had received a kidney transplant. Conclusion Renal involvement of HUV can be responsible for severe AKI, CKD and ESRD. It is not always associated with circulating anti-C1q antibodies. Kidney biopsy shows mostly MPGN or crescentic GN, with frequent C1q deposits in the glomeruli, tubules or arterioles.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease driven by complex interactions between genetics and environmental factors. SLE is characterised by breaking self-immune tolerance and autoantibody production that triggers inflammation and damage of multiple organs. Given the highly heterogeneous nature of SLE, the treatments currently used are still not satisfactory with considerable side effects, and the development of new therapies is a major health issue for better patient management. In this context, mouse models significantly contribute to our knowledge of the pathogenesis of SLE and are an invaluable tool for testing novel therapeutic targets. Here, we discuss the role of the most used SLE mouse models and their contribution to therapeutic improvement. Considering the complexity of developing targeted therapies for SLE, adjuvant therapies are also increasingly proposed. Indeed, murine and human studies have recently revealed that gut microbiota is a potential target and holds great promises for successful new SLE therapies. However, the mechanisms of gut microbiota dysbiosis in SLE remain unclear to date. In this review, we propose an inventory of existing studies investigating the relationship between gut microbiota dysbiosis and SLE to establish microbiome signature that may serve as a potential biomarker of the disease and its severity as well as a new potential therapy target. This approach may open new possibilities for early diagnosis, prevention and therapeutic perspectives of SLE based on gut microbiome.

Persistence of various symptoms in patients who have recovered from coronavirus disease 2019 (COVID-19) was recently defined as ‘long COVID’ or ‘post-COVID syndrome’ (PCS). This article reports a case of a 58-year-old woman who, although recovering from COVID-19, had novel and persistent symptoms including neurological complications that could not be explained by any cause other than PCS. In addition to a low inflammatory response, persistence of immunoglobulin G anticardiolipin autoantibody positivity and eosinopenia were found 1 year after acute COVID-19 infection, both of which have been defined previously as independent factors associated with the severity of COVID-19. The pathophysiological mechanism of PCS is unknown, but the possibility of persistence of the virus, especially in the nervous system, could be suggested with a post-infectious inflammatory or autoimmune reaction.

Abstract During the recent pandemic of SARS-CoV-2, and as a reaction to the worldwide shortage of surgical masks, several countries have introduced new types of masks named “community face coverings” (CoFC). To ensure the quality of such devices and their relevance to slow down the virus spreading, a quick reaction of the certification organisms was necessary to fix the minimal acceptable performances requirements. Moreover, many laboratories involved in the aerosol research field have been asked to perform tests in a quick time according to (CEN, 2020) proposed by the European committee for standardization. This specification imposes a minimal air permeability of 96 L m−2 s−1 for a 100 Pa pressure drop and a minimal filtration efficiency of 70% for 3 µm diameter particles. In the present article, an intercomparison of efficiencies and permeabilities measured by 3 laboratories has been performed. Results are in good agreement considering the heterogeneity of the material samples (within 27% in terms of filtration efficiency and less than 20% in terms of permeability). On this basis, an analysis of 233 materials made of woven, non-woven and mixed fibrous material has been done in terms of filtration efficiency and air permeability. For some of them, measurements have been performed for 0.2 µm, 1 µm and 3 µm particle diameters. As expected, no deterministic correlation could be determined to link these efficiencies to the permeability of the considered samples. However, a trend could be identified for woven and mixed materials with an increase of filtration efficiency when the air permeability decreases. The same exercise has been conducted to link the filtration efficiency measured at 3 µm to the one for lower diameters. Finally, a discussion on the kind of material that is the most relevant to manufacture CoFC supported by spectral filtration efficiency values (from 0.02 µm to 3 µm) is proposed.

European regulations require that the risk of an explosive atmosphere (ATEX) associated with the production of a flammable mist be assessed. In order to properly investigate mist flammability, a dedicated experimental tool was developed based on the standardized 20L explosion sphere and an additional Venturi injection system. As part of its design, the conditions of mist generation must be fully controlled. To complement the experimental approach, a computational fluid dynamic modeling (CFD) has been developed with an Euler-Lagrange method. It aimed at determining the homogeneity of the mist and the influence of the turbulence on the droplet size distribution (coalescence / fragmentation dynamics) as it greatly influences the mechanism of combustion. Based on previous studies and on industrial considerations, ethanol and kerosene were chosen. Modeling results were compared to the droplet size distribution determined by laser in-situ measurements and to the flow characteristics measured by particle image velocimetry. The CFD modeling confirmed the significant effect of the turbulence on the droplets coalescence and enabled to improve the understanding of the phenomenology related to the dispersion of a flammable mist into a 20L sphere. These results are consistent with the droplet particle distributions measured experimentally and show that CFD modeling can support further experimental developments so that it can constitute the basis for a new standard 20L explosion sphere for mists testing.

CD146 is an adhesion molecule essentially located in the vascular system, which has been described to play an important role in angiogenesis. A soluble form of CD146, called sCD146, is detected in the bloodstream and is known as an angiogenic factor. During placental development, CD146 is selectively expressed in extravillous trophoblasts. A growing body of evidence shows that CD146 and, in particular, sCD146, regulate extravillous trophoblasts migration and invasion both in vitro and in vivo. Hereby, we review expression and functions of CD146/sCD146 in the obstetrical field, mainly in pregnancy and in embryo implantation. We emphasized the relevance of quantifying sCD146 in the plasma of pregnant women or in embryo supernatant in the case of in vitro fertilization (IVF) to predict pathological pregnancy such as preeclampsia or implantation defect. This review will also shed light on some major results that led us to define CD146/sCD146 as a biomarker of placental development and paves the way toward identification of new therapeutic targets during implantation and pregnancy.

The dispersion conditions of the powders in a 20 L sphere are standardized to obtain reproducible results during explosion tests. However, experimental and numerical investigations have demonstrated that the dust cloud is often heterogeneous and that the nominal dust concentration can be significantly different from that reached at the sphere centre when ignition occurs. In order to improve the dust cloud homogeneity, and consequently, to enhance the repeatability of the method, six alternative dispersion nozzles were designed and tested. The time evolution of the turbulence and of the particle size distribution of the dust cloud were determined respectively by particle image velocimetry and in situ laser diffraction sensor. Euler-Lagrange simulations (Star-CCM+) and DEM simulations were performed to study the dust dispersion dynamics and to assess the importance of the dust fragmentation/agglomeration. Explosion tests were also run for some selected nozzles. Results showed that the turbulence level in the sphere was always lower with the 6 symmetrical nozzles in comparison to the standard rebound nozzle. A lowest turbulent kinetic energy during ignition generally leads to a more homogeneous dust dispersion and thus to a concentration profile at the sphere centre which is closer to the nominal dust concentration. Nevertheless, this decay also implies lower explosion severities and sometimes results in dust accumulation within the nozzles. The particle size distribution of the dust cloud varies from that of the original powder, but does not significantly changes after the first milliseconds of its dispersion within the sphere. Moreover, particle fragmentation was nearly identical for all the nozzles. Numerical and experimental results then demonstrated that the fragmentation is both caused by the injection through the injection valve and by the nozzle geometry. As a consequence, a specific attention must be paid to the injection procedure and to the ignition delay time, which is directly related to the turbulence of the dust cloud.

Venous thromboses have been associated with tuberculosis, but the relationship with circulating anticoagulant has not been studied yet. In a cohort of 48 patients with tuberculosis, 22.9% of them presented with venous thromboses significantly associated with dose dependent level of antiphosphophatidyl-ethanolamine antibodies. Keywords: Tuberculosis, Deep vein thrombosis, Pulmonary embolism, Antiphospholipds antibodies, Mycobacterium tuberculosis, Anti-phosphatidylethanolamine antibodies

Background: The detection of additional autoantibodies is of great concern in systemic sclerosis (SSc) when those included in the ACR/EULAR classification are negative. In this context, the interest of antifibrillarin (anti-U3RNP) autoantibodies (AFAs) in the routine evaluation of SSc remains unclear. We aimed to assess the relevance of AFAs and their clinical association in SSc patients. Methods: In a multicenter observational retrospective study, we collected immunological and clinical data associated with AFA positivity in SSc (n = 42) and non-SSc patients (n = 13). Patients with SSc negative for AFAs (n = 83) were considered as a control group. AFAs were detected by indirect immunofluorescence (IIF) using HEp-2 cells, EliA or immunoblot techniques. Results: We confirmed a typical nuclear IIF pattern and showed that AFAs are mostly exclusive towards SSc conventional autoantibodies. Although also observed in non-SSc patients, high levels of AFAs with the ELiA technique allowed the diagnosis of SSc. Compared to AFA-negative SSc patients, AFA-positive SSc patients more frequently exhibited visceral involvements. They more frequently suffered from the diffuse cutaneous form and had a higher global severity of the disease. Conclusions: We demonstrate the usefulness of quantifying AFAs in the immunological exploration of SSc, especially when patients are seronegative for SSc conventional autoantibodies and display a typical IIF pattern. AFAs might constitute an interesting marker of SSc severity.

CD146 is a cell adhesion molecule expressed on endothelial cells, as well as on other cells such as mesenchymal stem cells and Th17 lymphocytes. This protein also exists in a soluble form, whereby it can be detected in biological fluids, including the serum or the cerebrospinal fluid (CSF). Some studies have highlighted the significance of CD146 and its soluble form in angiogenesis and inflammation, having been shown to contribute to the pathogenesis of many inflammatory autoimmune diseases, such as systemic sclerosis, mellitus diabetes, rheumatoid arthritis, inflammatory bowel diseases, and multiple sclerosis. In this review, we will focus on how CD146 and sCD146 contribute to the pathogenesis of the aforementioned autoimmune diseases and discuss the relevance of considering it as a biomarker in these pathologies.

The fundamental role of cell adhesion molecules in mediating various biological processes as angiogenesis has been well-documented. CD146, an adhesion molecule of the immunoglobulin superfamily, and its soluble form, constitute major players in both physiological and pathological angiogenesis. A growing body of evidence shows soluble CD146 to be significantly elevated in the serum or interstitial fluid of patients with pathologies related to deregulated angiogenesis, as autoimmune diseases, obstetric and ocular pathologies, and cancers. To block the undesirable effects of this molecule, therapeutic antibodies have been developed. Herein, we review the multifaceted functions of CD146 in physiological and pathological angiogenesis and summarize the interest of using monoclonal antibodies for therapeutic purposes.

Although progress was made in in vitro fertilization (IVF) techniques, the majority of embryos transferred fail to implant. Morphology embryo scoring is the standard procedure for most of IVF centres for choosing the best embryo, but remains limited since even the embryos classified as "top quality" may not implant. As it has been shown that i) CD146 is involved in embryo implantation and ii) membrane form is shed to generate soluble CD146 (sCD146), we propose that sCD146 in embryo supernatants may constitute a new biomarker of embryo selection. Immunocytochemical staining showed expression of CD146 in early embryo stages and sCD146 was detected by ELISA and Western-blot in embryo supernatants from D2. We retrospectively studied 126 couples who underwent IVF attempt. The embryo culture medium from each transferred embryo (n = 222) was collected for measurement of sCD146 by ELISA. Significantly higher sCD146 concentrations were present in embryo supernatants that did not implant (n = 185) as compared to those that successfully implanted (n = 37) (1310 +/- 1152 pg.mL-1 vs. 845+/- 1173 pg.mL-1, p = 0.024). Sensitivity analysis performed on single embryo transfers (n = 71) confirmed this association (p = 0.0054). The computed ROC curve established that the optimal sCD146 concentration for embryo implantation is under 1164 pg.mL-1 (sensitivity: 76%, specificity: 48%, PPV: 25% and NPV: 92%). Over this sCD146 threshold, the implantation rate was significantly lower (9% with sCD146 levels >1164 pg.ml-1 vs. 22% with sCD146 levels ≤ 1164 pg.mL-1, p = 0.01). Among the embryos preselected by morphologic scoring, sCD146 determination could allow a better selection of the embryo(s), thus improving the success of elective single embryo transfer. This study establishes the proof of concept for the use of sCD146 as a biomarker for IVF by excluding the embryo with the highest sCD146 level. A multicentre prospective study will now be necessary to further establish its use in clinical practice.

The experimental characterization of combustible dusts is usually associated to several uncertainty factors that rely on the physical properties of the powder and the operating conditions of the standardized tests (e.g. the ignition time, the dispersion nozzle and pressure). These parameters affect the repeatability of the flammability tests and the precision, or even the accuracy, of their results. In this regard, an explanatory study has been focused on the description of the dispersion process of a combustible dust inside a 20-liters sphere by joining two complementary approaches. At first, a Computational Fluid Dynamics simulation (CFD – Star CCM+) based on an Euler-Lagrange scheme was set according to the geometry and operating parameters that have been established for this equipment through international standards. The predictive results that were determined with this approach were compared with a Particle Image Velocimetry analysis and in-situ measurements. Explosions tests were also performed to validate our analyses. The results showed how the particle size distribution of the dust affects its dispersion trajectories inside the sphere due to the inertial effects and the drag force exerted by the fluid. Furthermore, the analysis of the velocity field and turbulence intensity reveals that the dispersion process can be divided into three different stages, each related to a different repeatability. Tests performed with the classical rebound nozzle have clearly evidenced their effect on the internal distribution of the gas flow and the segregation levels of the disperse dust. These facts suggest that the homogeneity assumption that is usually considered for the dust dispersion is obviously invalid. As a consequence, the operating conditions should be adapted to the physical properties of each combustible powder in order to improve both tests accuracy and precision. For this purpose, CFD simulations have been proven to be a useful tool to identify the most suitable conditions to perform this analysis and obtain the most conservative information about the reactivity of the dust as well.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of multiple autoantibodies. Antibodies against Ficolin-3 were previously identified in the sera of some SLE patients, but their prevalence and significance have not been yet investigated. The aims of this study were to determine the prevalence of anti-ficolin-3 antibodies among SLE patients and to investigate their potential as diagnostic and/or prognostic biomarkers in SLE. In this retrospective study, sera from SLE patients (n = 165) were selected from a preexisting declared biological collection. Samples from healthy controls (n = 48) were matched with SLE sera. Disease activity was determined according to the SLEDAI score. Anti-ficolin-3, anti-dsDNA and anti-C1q antibodies levels were measured in sera by ELISA. First, a highly significant difference was found in the anti-ficolin-3 levels between SLE patients and healthy subjects. Anti-ficolin-3 antibodies were detected as positive in 56 of 165 (34%) SLE patients. The titer of anti-ficolin-3 antibodies was correlated with the SLEDAI score (r = 0.38, p

In a pilot ProtoArray analysis, we identified 6 proteins out of 9483 recognized by autoantibodies (AAb) from patients with systemic sclerosis (SSc). We further investigated the 6 candidates by ELISA on hundreds of controls and patients, including patients with Systemic Lupus Erythematosus (SLE), known for high sera reactivity and overlapping AAb with SSc. Only 2 of the 6 candidates, Ephrin type-B receptor 2 (EphB2) and Three prime Histone mRNA EXonuclease 1 (THEX1), remained significantly recognized by sera samples from SSc compared to controls (healthy or with rheumatic diseases) with, respectively, 34% versus 14% (P = 2.10-4) and 60% versus 28% (P = 3.10-8). Above all, EphB2 and THEX1 revealed to be mainly recognized by SLE sera samples with respectively 56%, (P = 2.10-10) and 82% (P = 5.10-13). As anti-EphB2 and anti-THEX1 AAb were found in both diseases, an epitope mapping was realized on each protein to refine SSc and SLE diagnosis. A 15-mer peptide from EphB2 allowed to identify 35% of SLE sera samples (N = 48) versus only 5% of any other sera samples (N = 157), including SSc sera samples. AAb titers were significantly higher in SLE sera (P

With the emergence of biotherapies, accurate diagnosis in early arthritis is needed. At this time, there is no biological marker of psoriatic arthritis.To test whether antinuclear antibodies (ANA) can be used as a diagnostic tool in psoriatic arthritis (PsA), we evaluated the prevalence of ANA in biologic-naïve PsA patients and in healthy blood donors.232 patients from the Rheumatology department, St Marguerite's Hospital, Marseilles, who fulfilled the CASPAR criteria for PsA, underwent clinical and laboratory investigations. Antinuclear antibodies (ANA), anti-extractable nuclear antigen antibodies (ENA), rheumatoid factor (RF), anti-citrullinated protein antibodies (ACPA) were assayed. Ninety-one healthy blood donors were also tested.Detection of ANA by indirect immunofluorescence was significantly more frequent in sera from PsA patients than those from controls at serum dilution of 1:100 (57% compared with 40%, Odds Ratio (OR) 1.98 (1.2-3.4) p

CD146 (MCAM, MUC18, S-Endo1) is a transmembrane glycoprotein belonging to both CAM and mucin families. It exists as different splice variants and is cleaved from the membrane by metalloproteases to generate a soluble form. CD146 is expressed by numerous cancer cells as well as being one of the numerous proteins expressed by the vascular endothelium. It has also been identified on smooth muscle cells, pericytes, and some immune cells. This protein was initially described as an actor involved in tumor growth and metastatic dissemination processes. Some recent works highlighted the role of CD146 in angiogenesis. Interestingly, this knowledge allowed the development of therapeutic and diagnostic tools specifically targeting the different CD146 variants. The first anti-CD146 antibody designed to study the function of this molecule, MUC18, was described by the Pr. J.P. Jonhson in 1987. In this review, we will discuss the 30 following years of research focused on the detection, study, and blocking of this protein in physiological and pathological processes.

Introduction. Indirect immunofluorescence (IIF) is the gold standard method for the detection of antinuclear antibodies (ANA) which are essential markers for the diagnosis of systemic autoimmune rheumatic diseases. For the discrimination of positive and negative samples, we propose here an original approach named Immunofluorescence for Computed Antinuclear antibody Rational Evaluation (ICARE) based on the calculation of a fluorescence index (FI). Methods. We made comparison between FI and visual evaluations on 237 consecutive samples and on a cohort of 25 patients with SLE. Results. We obtained very good technical performance of FI (95% sensitivity, 98% specificity, and a kappa of 0.92), even in a subgroup of weakly positive samples. A significant correlation between quantification of FI and IIF ANA titers was found (Spearman's ρ=0.80, P