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Introduction: The global SARS-CoV-2 pandemic significantly altered lifestyles, access to healthcare, and social interactions, introducing unprecedented physical and psychological stress all over the world. This study explores the relationship between psychological stress, vitamin D (Vit-D) levels, and autoimmune connective tissue diseases, including systemic lupus erythematosus, systemic sclerosis, polymyositis, dermatomyositis, and rheumatoid arthritis., Methods: The analysis was based on over one million patient data points derived from anti-nuclear antibody (ANA) testing conducted both before and during the COVID-19 pandemic 2017-2021. In a subset of patients, longitudinal data were collected bi-yearly to yearly over 5-8 years using the same three-month criteria. The dataset was analyzed using GraphPad Prism9 using paired t-tests or ordinary one-way ANOVA with a significance threshold of p < 0.05 to ensure robust correlations between the variables., Results: Data indicated that Vit-D levels peaked between 2017 and 2019 before declining, while ANA data demonstrated a rise in autoimmune connective tissue disease cases during the pandemic, reaching a peak in 2021. A clear correlation was observed, with autoimmune disease incidence increasing as Vit-D levels decreased. In-depth case analysis revealed that declining Vit-D levels preceded higher ANA titers and increased autoimmune disease severity, whereas improvements in Vit-D levels were associated with reduced ANA titers and less severe disease manifestations., Conclusions: The findings suggest that maintaining mental health and ensuring adequate Vit-D supplementation could be essential strategies for mitigating autoimmune disease risks and maintaining immune stability, particularly in pandemic scenarios. Clinically, these results underscore the need for early interventions targeting both psychological well-being and Vit-D levels to reduce the burden of autoimmune diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Alobaid and Alqabandi.)

Objective: Autoantibodies and autoimmune diseases after SARS-CoV-2 infection are widely reported. Given evolving variants, milder infections, and increasing population vaccination, we hypothesized that SARS-CoV-2 infection earlier in the pandemic would be associated with more autoimmune connective tissue disease (CTD) symptoms and immunologic abnormalities., Methods: Patients ≥18 years old with COVID-19 3/1/2020-8/15/2022 completed the CTD Screening Questionnaire and were tested for 27 autoimmune serologies, SARS-CoV-2 serologies, cell-bound complement activation products (CB-CAPs), and T and B lymphocyte immunophenotypes by flow cytometry. We assessed relationships between symptoms, serologies, and immunophenotypes in earlier (3/1/2020-1/31/2021) vs. later (2/1/2021-8/15/2022) periods, with different predominating SARS-CoV-2 viruses., Results: 57 subjects had earlier and 23 had later pandemic COVID-19. 35 % of earlier vs. 17 % of later pandemic patients had CTD symptoms (p 0.18). More patients were antinuclear antibody (ANA) positive (44 % vs. 13 %, p 0.01) and had lupus anticoagulant (11 % vs. 4 %, p 0.67). After adjustment for age, race, and sex, earlier (vs. later) COVID-19 was associated with increased ANA positivity (OR 4.60, 95%CI 1.17, 18.15). No subjects had positive CB-CAPs. T and B cell immunophenotypes and SARS-CoV-2 serologies did not differ by group. In heatmap analyses, higher autoantibody variety was seen among those with infection in the early pandemic., Conclusion: In this sample, having COVID-19 infection in the earlier (pre-2/1/2021) vs. later pandemic was associated with more CTD symptoms, ANA positivity, and autoantibody reactivities. Earlier SARS-CoV-2 variants circulating in a less vaccinated population with less natural immunity may have been more immunogenic., Competing Interests: Declaration of competing interest Roberta Alexander is a former employee and shareholder of Exagen Inc. May Choi is the Associate Director of MitogenDx. Dr. Sparks has received research support from Bristol Myers Squibb and performed consultancy for AbbVie, Amgen, Boehringer Ingelheim, Bristol Myers Squibb, Gilead, Inova Diagnostics, Janssen, Optum, Pfizer, ReCor, Sobi, and UCB unrelated to this work. Dr. Solomon receives salary support through unrelated research contracts to Brigham and Women's Hospital from CorEvitas, Janssen, and Novartis. Dr. Costenbader acknowledges the following: Amgen (Consultant, Grant/Research Support); AstraZeneca (Grant/Research Support); Bristol-Myers Squibb (BMS) (Consultant); Cabaletta Bio (Consultant); Eli Lilly (Consultant); Exagen Inc. (Grant/Research Support); Gilead (Grant/Research Support); GlaxoSmithKline (GSK) (Consultant, Grant/Research Support); Janssen (Consultant, Grant/Research Support). The funders had no role in the decision to publish or preparation of this manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of Harvard University, its affiliated academic health care centers, or the National Institutes of Health., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Whole-exome sequencing of two unrelated kindreds with systemic autoimmune disease featuring antinuclear antibodies with IgG4 elevation uncovered an identical ultrarare heterozygous TNIP1 Q333P variant segregating with disease. Mice with the orthologous Q346P variant developed antinuclear autoantibodies, salivary gland inflammation, elevated IgG2c, spontaneous germinal centers and expansion of age-associated B cells, plasma cells and follicular and extrafollicular helper T cells. B cell phenotypes were cell-autonomous and rescued by ablation of Toll-like receptor 7 (TLR7) or MyD88. The variant increased interferon-β without altering nuclear factor kappa-light-chain-enhancer of activated B cells signaling, and impaired MyD88 and IRAK1 recruitment to autophagosomes. Additionally, the Q333P variant impaired TNIP1 localization to damaged mitochondria and mitophagosome formation. Damaged mitochondria were abundant in the salivary epithelial cells of Tnip1 Q346P mice. These findings suggest that TNIP1-mediated autoimmunity may be a consequence of increased TLR7 signaling due to impaired recruitment of downstream signaling molecules and damaged mitochondria to autophagosomes and may thus respond to TLR7-targeted therapeutics., (© 2024. The Author(s).)

The diagnosis of systemic autoimmune diseases (SAID) is challenging, due to overlapping features with other non-immune disorders. Anti-nuclear antibodies (ANA)/anti-cellular antibodies are the sensitive screening tests but anti-double-stranded-deoxyribonucleic acid-antibody (anti-ds-DNA) and ANA-specific antibodies are specific for SAID. We aimed to look at ANA-specific antibodies in our patients and correlated them with ANA patterns, anti-ds-DNA, and clinical diagnosis for proper interpretation and better patient management cost-effectively. A retrospective data analysis of 641 patients was done (1st of February 2019 to 31st of July 2021) who were tested for ANA-specific antibodies at the Immunology Department of Indus Hospital and Health Network. ANA and anti-ds-DNA results and clinical diagnosis were also analyzed for ANA-specific antibody-positive patients. Descriptive data were presented in mean ± standard deviation and frequency percentages whereas inferential data were analyzed with a chi-square test for association between ANA-specific antibodies status, ANA, anti-ds-DNA, and clinical features. ANA-specific antibodies test revealed positivity for at least one autoantibody in 245 (38.2%) patients. Of these, ANA was tested in 206 patients reactive for ANA-specific antibodies and found positive in 195 (95%) as compared to negative (< 0.001). Speckled and homogenous were predominant ANA patterns in ANA-specific antibody-positives (56% and 42% respectively). Multiple ANA patterns were found in 18 patients most commonly with systemic lupus erythematosus (SLE) and mixed connective tissue disorder (MCTD). Anti-SSA were the most common ANA-specific antibodies (50%) and were mostly found in sera with speckled (61/97) and homogenous (38/97) patterns and associated mostly with SLE (48%) and Sjogren's syndrome (86%). Among ANA-negative patients, anti-SSA were the most common antibodies (n = 5). Anti-ds-DNA was found in 66% of SLE patients along with another ANA-specific antibody. This study showed that testing for ANA-specific antibodies cannot be gated on ANA patterns. Also, there is a redundancy of these antibodies with various clinical diagnoses. Moreover, they are useful in making a diagnosis in ANA-negative patients as well with clinical suspicion., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: We investigated the autoantibody (autoAb) profiles in ANA+ individuals lacking systemic autoimmune rheumatic disease (SARD) and early SARD patients to determine the key differences between these groups and identify factors that are associated with an increased risk of symptomatic progression within the next 2 years in ANA+ individuals., Methods: Using custom antigen (Ag) microarrays, 144 IgM and IgG autoAbs were surveyed in 84 asymptomatic and 123 symptomatic (48 UCTD and 75 SARD patients) ANA+ individuals. AutoAbs were compared in ANA+ individuals lacking a SARD diagnosis with ≥2 years follow-up (n = 52), including all those who demonstrated progression (n = 14) during this period, with changes over time assessed in a representative subset., Results: We show that ANA+ individuals have autoAb to many self-Ags that are not being captured by current screening techniques and very high levels of these autoAbs are predominantly restricted to early SARD patients, with SLE patients displaying reactivity to many more autoAgs than the other groups. In general, the symptoms that developed in progressors mirrored those seen in SARD patients with similar patterns of autoAbs. Only anti-Ro52 Abs were found to predict progression (positive predictive value 46%, negative predictive value 89%). Surprisingly, over 2 years of follow-up the levels of autoAbs remained remarkably stable regardless of whether individuals progressed or not., Conclusion: Our findings strongly argue that development of assays with an expanded set of auto-Ags and enhanced dynamic range would improve the diagnostic and prognostic ability of autoAb testing., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: The indirect immunofluorescence assay on HEp-2 cells (HEp-2/IFA) is used worldwide for screening for autoantibodies to cellular antigens. Cell culture and fixation methods influence the cell distribution of autoantigens and the preservation of epitopes. Therefore, discrepancy of results obtained using different HEp-2/IFA kits (interkit nonreproducibility) is a common phenomenon in the clinical laboratory routine., Objective: This study evaluated the interkit nonreproducibility of HEp-2/IFA results using samples from patients with systemic autoimmune disease (SAD), nonautoimmune diseases (NAD), and healthy blood donors (HBD)., Methods: Serum from 275 SAD patients, 293 NAD patients, and 300 HBD were processed at 1:80 dilution using four HEp-2 kits according to the manufacturers' instructions. Interkit reproducibility was determined for positive/negative results and patterns. The agreement of positive/negative results among kits for each sample was determined as the reactivity agreement score (RAS). The pattern reproducibility score (PRS) in each sample was calculated as a function of the number of kits showing equivalent patterns. Qualitative variables and ordinal variables were analyzed by the Chi-square and Mann-Whitney U tests, respectively., Results: A total of 402 samples were nonreactive in all kits and were considered devoid of autoantibodies. Further analysis included the 466 reactive samples (238 SAD, 119 NAD, 109 HBD). Reactivity to the nucleus had the highest interkit reproducibility (RAS = 83.6), followed by the metaphase plate (RAS = 78.9), cytoplasm (RAS = 77.4), and nucleolus (RAS = 72.4). Interkit reproducibility was higher in SAD (RAS = 78.0) than in NAD (RAS = 70.6) and HBD (RAS = 71.3) groups. Samples with strong reactivity (++++/4 and +++/4) had higher interkit reproducibility than those with weak reactivity (+/4). In the SAD group, RAS for nuclear reactivity was 87.5% for strongly reactive samples as opposed to 4.4% for weakly reactive samples, and the same was observed for NAD and HBD samples. The most robust patterns were the centromere AC-3 (PRS = 78.4), multiple nuclear dots AC-6 (PRS = 73.6), nuclear coarse speckled AC-5 (PRS = 71.3), nuclear homogeneous AC-1 (PRS = 67.9), and the reticular cytoplasmic AC-21 (PRS = 68.6)., Conclusion: Interkit nonreproducibility in HEp-2/IFA is prevalent and occurs with the highest frequency with weakly reactive samples. International initiatives with the engagement of in vitro diagnostic industry are encouraged to promote the harmonization of the properties and performance of HEp-2/IFA commercial kits., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Silva, Dellavance, Baldo, Rodrigues, Grecco, Prado, Agustinelli and Andrade.)

Results of the anti-nuclear antibodies-indirect immunofluorescence assay (anti-cell antibodies test) on HEp-2 cell substrates should be communicated to clinicians in a standardized way, adding value to laboratory findings and helping with critical clinical decisions. This paper proposes a test report based on the practices informed by 118 laboratories in 68 countries, with recommendations from the International Consensus on ANA Patterns (ICAP) group. Major focus is placed on the report format containing endpoint titers, immunofluorescence patterns together with anti-cell (AC) nomenclature, remarks on follow-up or reflex testing, and possible other autoantibody associations. ISO 15,189 directives were integrated into the test report. Special situations addressed include serum screening dilutions and endpoint titers, relevance of immunofluorescence patterns with special attention to cytoplasmic patterns, mixed and compound patterns, and how to report different titers corresponding to multiple patterns or autoantibodies in the same sample. This paper suggests a subtitle for the HEp-2-IIFA, namely anti-cell antibodies test, which could gradually substitute the original outdated ANA nomenclature. This ICAP pro forma report represents a further step in harmonizing the way relevant clinical information could be provided by laboratories., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Current literature regarding systemic autoimmune diseases in X-chromosome aneuploidies is scarce and limited to case reports. Our aim was to evaluate the frequency of anti-nuclear (ANAs), extractable nuclear (ENA), anti-double-stranded DNA (dsDNAs), anti-smooth muscle (ASMAs) and anti-mitochondrial (AMAs) antibodies in a large cohort of adults with Klinefelter's syndrome (KS, 47,XXY) and rare higher-grade sex chromosome aneuploidies (HGAs) for the first time. Sera from 138 X-chromosome aneuploid patients [124 adult patients with 47,XXY KS and 14 patients with HGA (six children, eight adults)] and 50 age-matched 46,XY controls were recruited from the Sapienza University of Rome (2007-17) and tested for ANAs, ENAs, anti-dsDNAs, ASMAs and AMAs. Non-organ-specific immunoreactivity was found to be significantly higher in patients with 47,XXY KS (14%) than in the controls (2%, p = 0.002). Among all the antibodies investigated, only ANAs were observed significantly more frequently in patients with 47,XXY KS (12.1%) than in the controls (2%, p = 0.004). No anti-dsDNA immunoreactivity was found. Stratifying by testosterone replacement therapy (TRT), non-organ-specific autoantibody frequencies were higher in TRT-naive (p = 0.01) and TRT-treated groups than in controls. No patients with HGA were found positive for the various autoantibodies. Non-organ-specific autoantibodies were significantly present in 47,XXY adult patients. Conversely, HGAs did not appear to be target of non-organ-specific immunoreactivity, suggesting that KS and HGAs should be considered as two distinct conditions. The classification and diagnosis of systemic autoimmune diseases is frequently difficult. To support a correct clinical evaluation of KS disease and to prevent eventual secondary irreversible immune-mediated damages, we highlight the importance of screening for non-organ-specific autoimmunity in Klinefelter's syndrome., (© 2021 British Society for Immunology.)

Background: The diagnosis of systemic autoimmune rheumatic diseases (SARD) is based on the detection of serum antinuclear antibodies (ANA) for which indirect immunofluorescence (IIF) is the golden standard. New solid-phase immunoassays have been developed to be used alone or in combination with the detection of extractable antinuclear antibodies (ENA) to improve SARD diagnosis. The purpose of this study was to compare the clinical performances of different ANA screening methods alone or in combination with ENA screening methods for SARD diagnosis., Methods: A total of 323 patients were screened for ANA by IIF, EliA™ CTD Screen, and ELISA methods. Agreements were calculated between the methods. Then, EliA™ CTD Screen positive samples were screened for ENA by line immunoassay (LIA) and fluorescence enzyme immunoassay (FEIA)., Results: The diagnostic accuracy of EliA™ CTD Screen (79% sensitivity and 91% specificity) was better than that of ELISA or IIF. The combination of EliA™ CTD plus IIF had the highest sensitivity (93%). ENA determination revealed that Ro52 and Ro60 were the most prevalent specificities. The use of IIF alone was not able of detecting up to 36% of samples positive for Ro52, and 41% for Ro60., Conclusions: EliA™ CTD Screen has a better diagnostic performance when compared to IIF and ELISA. The combined use of EliA™ CTD Screen and IIF clearly improves the rate and accuracy of SARD diagnosis. The use of EliA™ CTD Screen as first-line screening technique allows the detection of antibodies, which could not be detected by IIF alone., (© 2021 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Introduction: The clinical significance of common antinuclear antibody (ANA) patterns, such as nuclear homogenous and nuclear speckled patterns with their corresponding specific antibodies, has already been established. However, the clinical relevance of these uncommon ANA patterns have not been well elucidated and these patterns are therefore not reported by most clinical laboratories. We herein report some retrospective data analysis linking patients' clinical status to several uncommon ANA patterns., Methods: We retrieved and assessed the patient records for ANA reports generated in our hospital over a period of two years. All testing had been performed using the gold standard Indirect Immunofluorescence Assay., Results: Records of 1235 consecutive patients tested for ANA were reviewed. ANA was positive in 330 of these patients with 6.39% found to have uncommon nuclear, cytoplasmic or mitotic sub-patterns. The mitotic spindle (0.89%), cytoplasmic anti-mitochondrial antibodies (0.80%), followed by discrete nuclear dots-multiple (0.72%) were the dominating patterns, with a higher prevalence in females than in males. Systemic lupus erythematosus and rheumatoid arthritis were the two most common autoimmune disorders associated with mitotic spindle fibers and nuclear centromere and nuclear large/coarse speckled ANA patterns., Conclusion: The prevalence of these relatively uncommon ANA patterns was higher than expected. Further evaluation of these patterns along with their corresponding antibodies and their clinical utility must be encouraged. We trust this endeavour will provide diagnostic information in autoimmune and other disease conditions., (Copyright © 2021 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Abstract: Previous studies from various countries have reported anti-dense fine speckled pattern (DFS)70 antibody prevalence but few studies have been from Asia. We investigated the prevalence of anti-DFS70 autoantibodies in a Japanese cohort of healthy individuals (HI) and patients with antinuclear antibody-associated autoimmune rheumatic diseases (AARD).Enzyme-linked immunosorbent assay and indirect immunofluorescence were performed using samples from 250 HI and 276 AARD patients.The overall anti-DFS70 antibody prevalence in HI was 16.4%, with 12.8% for males and 20.0% for females (sex difference; P = .12). In AARD patients, the anti-DFS70 antibody prevalence in systemic lupus erythematosus, mixed connective tissue disease, systemic sclerosis, dermatomyositis and polymyositis (DM/PM), Sjögren syndrome, and rheumatoid arthritis (RA) was 22.1%, 14.3%, 14.3%, 3.0%, 21.3%, and 18.1%, respectively (no significant difference between AARD patients except DM/PM and HI). The prevalence of isolated anti-DFS70 antibody in HI and all AARD patients excluding RA was 14.8% (37/250) and 4.4% (9/204), respectively (P  < .01 vs HI). Among anti-DFS70 antibody-positive cases, 63.4% (26/41) were DFS pattern by IIF and 23.5% (8/34) were HI and AARD patients excluding RA, respectively.The anti-DFS70 antibody prevalence in HI and AARD patients in Japan was similar. Furthermore, the anti-DFS70 antibody prevalence in HI and AARD in Japan is higher than in HI and AARD in regions other than Asia. This makes AARD differential diagnosis by antinuclear antibody screening difficult., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Anti-Ro60 is one of the most common and clinically important serum autoantibodies that has a number of diagnostic and predictive capabilities. Most diagnostic laboratories report this simply as a qualitative positive/negative result. The objective of this study was to examine the clinical and serological relevance of a novel subset of anti-Ro60 in patients who display low levels of anti-Ro60 (anti-Ro60 low ). We retrospectively identified anti-Ro60 sera during a 12-month period at a major immunopathology diagnostic laboratory in Australia. These all were anti-Ro60-precipitin-positive on the diagnostic gold standard counter-immuno-electrophoresis (CIEP). Lineblot immunoassay was used to stratify patients into either anti-Ro60 low or anti-Ro60 high subsets. We compared the medical and laboratory parameters associated with each group. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry techniques were used to analyse the serological and molecular basis behind the two subsets. Anti-Ro60 low patients displayed less serological activity than anti-Ro60 high patients with less intermolecular spreading, hypergammaglobulinaemia and less tendency to undergo anti-Ro60 isotype-switching than anti-Ro60 high patients. Mass spectrometric typing of the anti-Ro60 low subset showed restricted variable heavy chain subfamily usage and amino acid point mutations. This subset also displayed clinical relevance, being present in a number of patients with systemic autoimmune rheumatic diseases (SARD). We identify a novel anti-Ro60 low patient subset that is distinct from anti-Ro60 high patients serologically and molecularly. It is not clear whether they arise from common or separate origins; however, they probably have different developmental pathways to account for the stark difference in immunological maturity. We hence demonstrate significance to anti-Ro60 low and justify accurate detection in the diagnostic laboratory., (© 2020 British Society for Immunology.)

Antinuclear antibodies (ANAs) are valuable laboratory markers to screen for and support the diagnosis of various rheumatic diseases (known as ANA-associated rheumatic diseases). The importance of ANA testing has been reinforced by the inclusion of ANA positivity as an entry criterion in the 2019 systemic lupus erythematosus classification criteria. In addition, specific ANAs (such as antibodies to Sm, double-stranded DNA (dsDNA), SSA/Ro60, U1RNP, topoisomerase I, centromere protein B (CENPB), RNA polymerase III and Jo1) are included in classification criteria for other rheumatic diseases. A number of techniques are available for detecting antibodies to a selection of clinically relevant antigens (such as indirect immunofluorescence and solid phase assays). In this Review, we discuss the advantages and limitations of these techniques, as well as the clinical relevance of the differences between the techniques, to provide guidance in understanding and interpreting ANA test results. Such understanding not only necessitates insight into the sensitivity and specificity of each assay, but also into the importance of the disease context and antibody level. We also highlight the value of titre-specific information (such as likelihood ratios).

Though the exact etiology of autoimmune diseases still remains not completely known, there are various factors which are known to contribute to be trigger of autoimmune diseases. Viral infection is known to be among the other. It is known as the infection from severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) can be an autoimmune trigger, so, we suppose that SARS-Coronavirus (SARS-CoV-2) could be as well. Several authors have highlighted the temporal consequence between SARS-CoV-2 and autoimmune diseases. In this case report we described a patient admitted for COVID-19 pneumonia with completely negative autoimmunity at admission who developed major pulmonary interstitial disease. During the hospitalization the weaning difficulties from oxygen led us to the repetition of autoimmunity pattern which became positive (both during hospitalization then after two months from dismission) with marked positivity for specific antibodies for myositis even after the patient's infectious healing. In the follow-up, the patient continued to have asthenia and muscle weakness despite steroid therapy. She is still in follow-up and will be further evaluated over time. Can we therefore think that in this case the development of autoimmunity can persist beyond the infectious phase and determine over time the development of a real autoimmune myositis?

The coronavirus disease-2019 (COVID-19) pandemic is likely to pose new challenges to the rheumatology community in the near and distant future. Some of the challenges, like the severity of COVID-19 among patients on immunosuppressive agents, are predictable and are being evaluated with great care and effort across the globe. A few others, such as atypical manifestations of COVID-19 mimicking rheumatic musculoskeletal diseases (RMDs) are being reported. Like in many other viral infections, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection can potentially lead to an array of rheumatological and autoimmune manifestations by molecular mimicry (cross-reacting epitope between the virus and the host), bystander killing (virus-specific CD8 + T cells migrating to the target tissues and exerting cytotoxicity), epitope spreading, viral persistence (polyclonal activation due to the constant presence of viral antigens driving immune-mediated injury) and formation of neutrophil extracellular traps. In addition, the myriad of antiviral drugs presently being tried in the treatment of COVID-19 can result in several rheumatic musculoskeletal adverse effects. In this review, we have addressed the possible spectrum and mechanisms of various autoimmune and rheumatic musculoskeletal manifestations that can be precipitated by COVID-19 infection, its therapy, and the preventive strategies to contain the infection.

Assessing cardiac function and risk stratification in a fetal anti-Sjögren syndrome type A (SSA) or anti-Sjögren syndrome type B (SSB) complete atrioventricular block (CAVB) is challenging. We aimed to evaluate the cardiovascular profile score (CVP) and its components in surveillance of fetuses with autoimmune CAVB., Methods: Retrospective cohort review of CAVB pregnancies, excluding fetuses with significant cardiac anomalies., Results: CAVBs are in 17 fetuses, diagnosed at mean gestational age of 23 ± 5 weeks. Overall mortality is 18%: 1 termination, 1 fetal demise (intrauterine fetal demise [IUFD]), and 1 postnatal death. Both mortalities had intrauterine growth restriction; IUFD had placental infarction. Presenting CVP 8.7 ± 1. No fetus had CVP <7; the score correlated with increased risk of perinatal death. The 2 mortalities had initial CVP scores of 8 and 9; both increased to 10 on subsequent exams. 30% of fetuses had low middle cerebral artery pulsatility (MCA-PI) on the last study. All had high umbilical artery pulsatility (UA-PI) throughout gestation. The 2 deaths had the lowest MCA-PI., Conclusion: Despite low heart rates, high CVP scores in our cohort remained high and were not predictive of mortality. Abnormalities in MCA flow reflects fetal cerebral vasodilation that may indicate altered hemodynamics and be predictive of outcomes, but data is limited. Abnormal umbilical artery (UA) flow suggests that perinatal mortality may also be related to placental disease., (© 2020 John Wiley & Sons, Ltd.)

The performance of the automated indirect immunofluorescence system was compared with the manual method for detection of antinuclear antibodies (ANA) from 354 clinical serum samples. We compared the results (negative or positive), ANA patterns, and titers for the two methods. The coincidence rates for ANA positive and negative samples were 93.4% and 98.7%, respectively. The coincidence rates for single patterns, mixed patterns, and the final titers were 85.1%, 87.6%, and 87.6%, respectively. The homogeneous, speckled, cytoplasmic, centromere, multiple nuclear dots, and nucleolar pattern coincidence rates were 79.3%, 83.0%, 87.8%, 72.7%, 50%, and 56.3%, respectively. The automated indirect immunofluorescence system had acceptable accuracy for detection of ANA., (© 2020 by the Association of Clinical Scientists, Inc.)

Introduction: ndirect immunofluorescence assay (IFA) using HEp-2 as substrate plays a consolidate role for the detection and measurement of ANA, which is currently considered as the reference method for detection. Manual operation is still very common in China, therefore, the need of standardization and automation for ANA-IFA detecting has been highlighted., Objective: The current multi-center study is aimed to evaluate if HELIOS (AESKU Diagnostics, Wendelsheim, Germany) contributes to comparability of ANA screening results among different labs，and establish application specification of HELIOS for standardization of ANA detection., Methods: ANA detection by manual IFA method and HELIOS on 230 clinical serum samples in eight laboratories. The performance to discriminate positive/negative screening results, endpoint titer estimation and pattern recognition were evaluated in HELIOS and manual visual., Results: The positive coincident rate for ANA detection by manual IFA ranges from 87.7% to 97.8%, the negative coincidence rate ranges from 68.8% to 100%, the correctly estimated titer evaluation were 80 to 171 cases, the correct pattern in 146 to 161 cases, respectively. The positive coincident rate of HELIOS for ANA detection ranges from 91.2% to 97.7%, the negative coincidence rate ranges from 96.5% to 100%, the correctly estimated titer evaluation were 145 to 157 cases, the correct pattern in 123 to 140 cases, respectively., Conclusion: HELIOS could provide accurate diagnostic results, this include not only positive/negative results, but also endpoint titer, common patterns. The application of this system can help to promote standardization of ANA detection., (Copyright © 2019. Published by Elsevier B.V.)

Primary Biliary Cholangitis (PBC) is an uncommon, chronic, cholangiopathy of autoimmune origin and unknown etiology characterized by positive anti-mitochondrial autoantibodies (AMA), female preponderance and progression to cirrhosis if left untreated. The diagnosis is based on AMA- or PBC-specific anti-nuclear antibody (ANA)-positivity in the presence of a cholestatic biochemical profile, histologic confirmation being mandatory only in seronegative cases. First-line treatment is ursodeoxycholic acid (UDCA), which is effective in preventing disease progression in about two thirds of the patients. The only approved second-line treatment is obeticholic acid. This article summarizes the most relevant conclusions of a meeting held in Lugano, Switzerland, from September 23rd-25th 2018, gathering basic and clinical scientists with various background from around the world to discuss the latest advances in PBC research. The meeting was dedicated to Ian Mackay, pioneer in the field of autoimmune liver diseases. The role of liver histology needs to be reconsidered: liver pathology consistent with PBC in AMA-positive individuals without biochemical cholestasis is increasingly reported, raising the question as to whether biochemical cholestasis is a reliable disease marker for both clinical practice and trials. The urgent need for new biomarkers, including more accurate markers of cholestasis, was also widely discussed during the meeting. Moreover, new insights in interactions of bile acids with biliary epithelia in PBC provide solid evidence of a role for impaired epithelial protection against potentially toxic hydrophobic bile acids, raising the fundamental question as to whether this bile acid-induced epithelial damage is the cause or the consequence of the autoimmune attack to the biliary epithelium. Strategies are needed to identify difficult-to-treat patients at an early disease stage, when new therapeutic approaches targeting immunologic pathways, in addition to bile acid-based therapies, may be effective. In conclusion, using interdisciplinary approaches, groundbreaking advances can be expected before long in respect to our understanding of the etiopathogenesis of PBC, with the ultimate aim of improving its treatment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Autoantibodies to glycine and tryptophan-rich bodies (GWB) can be detected on routine antinuclear antibodies (ANA) testing and might have important disease associations. The aim of this study was to investigate the prevalence of anti-GWB antibodies identified on routine ANA testing, define their antigenic specificities and describe their clinical association. Anti-GWB antibodies were identified by distinct cytoplasmic staining pattern on all samples referred for ANA testing over a 6-month period. All positive anti-GWB samples were further tested on a multiplex addressable bead immunoassay (ALBIA) with known GWB antigens. Extractable nuclear antigens (ENA) were characterised by line immunoblot assay. Clinical details were collected retrospectively by contacting patients and the requesting clinicians. Eleven patients (7 females, 4 males) out of a total of 2136 positive ANAs requested on 11,265 samples had the classical GWB pattern (0.5%). The median age of patients was 66 years (range 39-92). There was no consistent disease association. Ten were confirmed to have distinct antigenic specificity for known GWB antigens. Ge-1/Hedls and RAP55 were the most common antigenic specificity targets [seen in 7 patients (64%) and in 5 patients (45%), respectively]. Ro52 was positive in 5/9 (56%) patients, SSB in 2/9 (22%) patients and Ro60 in 1/9 (11%) patient. The clinical association of anti-GWB antibodies is uncertain but might point towards autoimmune origin of certain non-specific musculoskeletal symptoms. The antigenic specificity of anti-GWB reactivity could point towards specific clinical associations: anti-RAP55 and Ge-1 in non-specific musculoskeletal conditions versus anti-GW182 in neurological diseases., (Crown Copyright © 2019. Published by Elsevier B.V. All rights reserved.)

Age-associated increases in antinuclear antibodies (ANA) in the general population are commonly noted but the mechanisms underlying this observation are unclear. This study aims to evaluate whether shorter peripheral blood mononuclear cell (PBMC) telomere length, a marker of more advanced biological age, is associated with ANA positivity prevalence and incidence in middle and older aged autoimmune disease-free individuals from the Baltimore Longitudinal Study of Aging (BLSA). Telomere length was measured by Southern Blot and categorized into tertiles. ANA was measured in a 1:80 and a 1:160 dilution of sera by immunofluorescence using HEp-2 cells (seropositive = 3 or 4). Multiple logistic regression was used to estimate the odds ratios and 95% confidence intervals of ANA positivity comparing the shorter tertiles of telomere length to the longest tertile for two cross-sectional points in time and then longitudinally to assess the association between shorter telomere length and incident ANA positivity. Cross-sectional analyses were adjusted for sex, race and BMI (N = 368 baseline, N = 370 follow-up) and longitudinal analyses were adjusted for sex, race, BMI and time between baseline and follow-up (N = 246). No statistically significant cross-sectional associations were observed at baseline or follow-up. Among those where ANA negative at baseline, individuals with shorter telomeres were more likely to be ANA positive at follow-up, an average 13 years later. Individuals with short telomeres at both time periods were more likely to be ANA positive. Findings suggest that ANA positivity in the general population may be indicative of immune dysfunction resulting from advanced cellular aging processes., (Published by Elsevier Ltd.)

Background: Chronic silica exposure can lead to silicosis, complicated or not by autoimmune diseases (AID). The pathophysiology of silica-induced AID remains not fully understood, especially immune mechanisms that may develop in patients without yet established silicosis. We conducted a prospective clinical study to analyze the impact of crystalline silica (CS) on T cell phenotype and regulatory T cells (Tregs) frequency, as well as on auto-antibodies development in non-silicotic workers exposed to CS. Methods: Workers with moderate to high exposure level to CS and aged between 30 and 60 years-old were considered for inclusion. Peripheral blood mononuclear cells were analyzed by flow cytometry. Auto-antibodies were screened in serum by immunofluorescence. Blood from 42 and 45 healthy subjects (HC) was used as control for T cell phenotype and serum analyses, respectively. Results: Among the 63 included workers exposed to CS, 55 had full data available and were analyzed. Ten were exposed to CS for <5 years, 18 for 5-10 years and 27 for more than 10 years. The frequency of Tregs (CD4 + CD25 + CD127 - FoxP3 + ) was significantly lower in CS exposed workers as compared to HC. We found an increased expression of the activation marker HLA-DR on T cells (CD3 + , CD4 + , and CD8 + ) of CS exposed workers as compared to HC. Tregs to activated T cells ratio was also lower in exposed subjects. In the latter, HLA-DR expression level and Tregs frequency were significantly associated with CS exposure duration. Serum autoantibody detection was significantly higher in CS exposed workers as compared to HC. Especially, among workers exposed more than 10 years, antinuclear antibodies and ANCA were detected in 44 and 22% among them, as compared to 5 and 2.5% in HC, respectively. Conclusion: This work shows that CS exposure is associated with a decrease of Tregs frequency, an increase of T cell activation status, and a tolerance breakdown against auto-antigens. These results show that alterations of the T cell compartment can be detected early over the course of CS exposure, preceding silicosis development or AID onset., (Copyright © 2019 Brilland, Beauvillain, Mazurkiewicz, Rucay, Roquelaure, Tabiasco, Vinatier, Riou, Jeannin, Renier, Subra and Augusto.)

Background: Rhombencephalitis (RE) is a serious condition of the brain with multiple etiologies. We report a unique case of recurrent, postpartum RE that is associated with positive anti-centromere antibody (ACA). A discussion of the case, current literature on autoimmune RE and related autoantibodies are reviewed., Case Presentation: A healthy 33-year-old Caucasian patient (gravida 2, para 2) had two episodes of progressive focal neurological deficits during postpartum periods. Signs and symptoms included right-sided dysmetria, adiadochokinesia, weakness, ataxia, and photophobia. MRI revealed rhombencephalitis involving the mesencephalon of the brainstem. Extensive and comprehensive investigations using blood and cerebrospinal fluid (CSF) were consistently positive only for ACA. The first episode was successfully treated with empiric antimicrobial agents and steroid. Given the negative infectious work up with the prior episode and the nearly identical clinical presentations, the second episode was treated with corticosteroid only. This led to complete resolution of her symptoms and reversal of the brain magnetic resonance imaging (MRI) lesions., Conclusion: To the author's knowledge, this is the first report of a primary autoimmune RE during postpartum period that is associated with ACA. Immunologic causes should be considered early with any encephalitis. Given the risk of recurrence, relapse, and neurologic deterioration, regular monitoring is recommended, especially for female patients of child-bearing age. Consistent with the current literature on autoimmune RE, steroid seems to be an effective treatment for ACA-associated RE.

Background: In China, the incidence of autoimmune diseases is gradually increasing. To decrease the misdiagnosis rate of autoimmune diseases, we conducted an epidemiological investigation about the presence of antinuclear antibody (ANA) in healthy populations and analyzed the clinical characteristics of healthy population with both high titer of ANA and positive anti-SSA and AMA-M2., Methods: Serum ANA titers were detected by indirect immunofluorescence (IIF), and other 15 types of ANA-specific antibodies were detected by line immunoassays., Results: In 25 110 individuals for routine examination, the positive rate of ANA titer >1:100 was 14.01%, of which the positive rate of female (19.05%) was higher than that of male (9.04%; P < 0.01). The positive rate of ANA titer >1:320 was 5.93%, of which the positive rate of female (8.68%) was higher than that of male (3.21%; P < 0.01). The specific antibodies were detected in 1489 of ANA-positive people with titer >1:320, and the top three detected antibodies were anti-Ro-52 (212), AMA-M2 (189), and anti-SSA (144). The abnormal rate of blood routine test, liver function test, and other clinical indicators in AMA-M2-positive population was significantly different from those in the control group. The abnormal rate of blood routine test, liver function test, and immune index in anti-SSA-positive population was higher than those in control group., Conclusion: There was a high prevalence of ANA positive in healthy population. To avoid misdiagnosis, those who had symptoms of abdominal discomfort, pruritus, or fatigue with abnormal results of blood routine and liver function test should be examined for ANA, AMA-M2, anti-SSA as early as possible., (© 2019 The Authors. Journal of Clinical Laboratory Analysis Published by Wiley Periodicals, Inc.)

Recent data suggest the implication of T, B and NK cells in the anti-synthetase syndrome (ASyS); nevertheless their role and activation states are poorly described. We performed deep immune-profiling using 37 markers on peripheral blood cells from 10 ASyS patients versus 17 healthy donors (HD) and 26 myositis control patients. We show decreased percentages of memory B cells in ASyS patients (mean ± SEM: ASyS = 13 ± 3%, HD = 37 ± 4% and 'myositis controls' = 32 ± 3), counterbalanced by increased percentages of naïve B cells. Interestingly, perifascicular infiltrations of memory B cells within muscle biopsies of ASyS patients suggest that they niche within the muscle., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Autoimmune cholangitis (AIC) was first reported in 1987 as a chronic cholestatic disease that occurs predominantly in middle-aged women and has a common clinical manifestations, biochemical abnormalities and pathological changes with primary biliary cholangitis (PBC). However, serum anti-mitochondrial antibodies (AMA) are negative, and ANA and/or smooth muscle antibody positive rates are higher. The treatment response and prognosis with ursodeoxycholic acid and steroids is poor, thus it needs to be treated with immunosuppressive agents. Presently, the exact pathological mechanism of AIC is still unclear, and there is no unified assertion that classifies it as a new autoimmune liver disease or AMA-negative PBC. This article reviews the worldwide published work on AIC and compares them with PBC.

Anti-SS-A antibodies are often sought for in autoimmune diseases diagnosis. Two different target proteins have actually been identified: Ro52 and Ro60. Clinical and immunological associations seem different depending on anti-Ro52 or anti-Ro60 antibodies presence. However, due to a heterogeneous presentation in the literature, some immunology laboratories in France have stopped providing anti-Ro52 antibody findings. We report here a new hospital study designed to determine the diagnostic utility of the separate detection of anti-Ro52 and anti-Ro60 antibodies. We conducted a retrospective, observational study, including every adult patient with positive antinuclear antibodies (ANA) tested in our immunology laboratory, and associated with anti-Ro52 and/or anti-Ro60 antibodies, between 2011 and 2014. Out of 13032 sera tested for ANA, 399 adults had antibodies to Ro52 and/or Ro60; 81.7% were female, with a mean age of 54.5 ± 17.0 years. Anti-Ro52 antibodies were found in 75.7% of the patients and anti-Ro60 antibodies in 56.9%. Among them, 43.1% were classified in the Ro52 + Ro60- group, 32.6% in the Ro52 + Ro60 + group and 24.3% in the Ro52-Ro60+ group. In the Ro52-Ro60+ group, systemic lupus was the most frequent diagnosis (48.5%), with a possible association with antiphospholipid antibodies (anti-cardiolipin antibodies: OR 2.5 (CI95 [1.0-5.0], p = 0.05) and lupus anticoagulant {OR 3.6 (CI95 [1.10-10.0] p = 0.02)}. In the Ro52+Ro60+, primary Sjögren Syndrome was the most likely (OR 4.2 95% CI [2.1-8.3] p < 10 -4 ), especially in patients Ro52+Ro60+La+. Patients with isolated anti-Ro52 had a wider variety of diseases associated, but among auto-immune diseases they were more prone to inflammatory myositis (OR 10.5 [1.4-81.7], p = 0.02) and inflammatory rheumatism (OR 4.6 [1.6-13.8], p = 0.006) in contrast to systemic lupus (OR 0.2 [0.1-0.3], p < 10 -4 ) or primary Sjögren's syndrome (OR 0.1 [0.06-0.2], p < 10 -4 ). We therefore suggest that, when anti-ENA antibodies are prescribed, it should include separate anti-Ro52 and anti-Ro60 antibodies determination. To go even further, we would like to suggest a change in ENA nomenclature to avoid confusion, abandoning the anti-SS-A label in favor of the anti-Ro52/TRIM21 or anti-Ro60 antibody for a clearer designation.

Several conditions have clinical and laboratory features that can mimic those present in Systemic Lupus Erythematosus (SLE). Some of these "SLE mimickers" are very common, such as rosacea which can be mistaken for the butterfly rash, while others such as Kikuchi disease, type-1 interferonopathies, Castleman's disease, prolidase deficiency, angioimmunoblastic T-cell lymphoma, Evans' syndrome in the context of primary immune deficiencies and the autoimmune lymphoproliferative syndrome are exceptionally uncommon. A proper diagnosis of SLE must therefore be based upon a complete medical history as well as on the adequate constellation of clinical or laboratory findings. While there is no single test that determines whether a patient has lupus or not, the search for auto-antibodies towards nuclear antigens is a key step in the diagnosis strategy, keeping in mind that ANAs are not specific for SLE. In case of persistent doubt, patients should be referred to reference centers with experience in the management of the disease., (Copyright © 2018. Published by Elsevier Masson SAS.)

Aim: Anti-centromere antibody (ACA) is often detected in patients with autoimmune diseases, including limited cutaneous systemic sclerosis (SSc), Sjögren's syndrome (SS), and primary biliary cholangitis (PBC). The association between autoimmune disease and ACA positivity remains unclear. We sought to clarify the clinical features of ACA-positive patients and their association with autoantibodies., Method: A total of 309 cases of a discrete-speckled pattern anti-nuclear antibody (ANA) test and/or positive ACA who visited our department were retrospectively enrolled. Clinical and immunological data were collected and statistically analyzed., Result: A proportion of second and/or third ANA patterns were speckled (16%), homogenous (7%), cytoplasmic (3%) and/or nucleolar (3%). Of the 309 patients, 186 had Raynaud's phenomenon, 149 had sclerodactyly, and 162 had oral and/or ocular dryness. A total of 214 patients were classified into 17 autoimmune diseases based on their symptoms at the initial visit, while the other 95 patients did not meet any criteria. Most of the 214 patients were diagnosed with SSc and/or SS; 25 and 22 additional patients were diagnosed with rheumatoid arthritis and PBC, respectively. Higher titers of immunoglobulins were observed in patients diagnosed with autoimmune disease compared to patients without a diagnosis. The mean observation period was 80 months. Three additional patients received interim diagnoses based on new symptoms or organ involvement. In the other patients, the diagnosis made at the first visit was not changed over the observation period., Conclusion: Our study confirmed that many ACA-positive cases can be classified into an autoimmune disease type on presentation., (© 2018 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.)

Background: Diagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA + individuals to determine whether they share any of the cellular immunologic features seen in SARD., Methods: Healthy ANA - controls and ANA + (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString., Results: A number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA + subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA + subjects and subjects with UCTD clustered  with patients with early SARD, rather than ANA - healthy controls., Conclusions: ANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD.

Background Data on the clinical relevance of borderline results of solid-phase assays in the screening for antinuclear antibodies (ANA) are sparse. This study aimed to determine the clinical significance of borderline results of the Elia CTD Screen (ECS; Phadia/Thermo Fisher Scientific, Freiburg, Germany), a fluoroenzymeimmunoassay incorporating 17 recombinant human nuclear antigens. Methods We retrospectively examined the medical records of 143 subjects with borderline ECS results for ANA-associated autoimmune disorders (AASARD) and the association with the results of indirect immunofluorescence (IIF) and confirmatory assays for ANA. Results AASARD were diagnosed in 10 patients (7%) with systemic lupus erythematosus (n=5; four patients were prediagnosed and in clinical remission), polymyositis overlap syndromes (n=2), scleroderma, Raynaud's syndrome and undetermined connective tissue disease (each n=1). Most frequently, homogeneous and nucleolar IIF patterns were found. Positive ANA subsets were observed in three patients. Furthermore, four patients were diagnosed with autoimmune liver diseases and yielded positive IIF in three and positive confirmatory assays in all cases. Taken together, 129 subjects had no AASARD. Within this group, 43 patients were IIF positive and most frequently showed speckled, unspecific nucleolar and only rarely homogeneous patterns. Positive ANA subsets were found in low concentrations near to the upper reference range in 18 subjects. Conclusions AASARD were observed in 7% of the subjects with borderline ECS and showed homogeneous or nucleolar IIF patterns in the majority of these cases. Our findings suggest that borderline results of the ECS can be clinically relevant and support the concept of a parallel or sequential screening for ANA by both ECS and IIF.

Patients with Interstitial Lung Disease (ILD) without a definitive diagnosis of connective tissue diseases (CTD) were historically described as Undifferentiated Connective Tissue Disease (UCTD-ILD). Recently a new classification, Interstitial Pneumonia with Autoimmune Features (IPAF), has been proposed. Aim of this study was to describe the prevalence, clinical characteristics and prognostic factors of UCTD and IPAF subjects in a cohort of Non-Specific Interstitial Pneumonia (NSIP) patients. This retrospective, observational study enrolled 102 adult patients characterized by NSIP pattern on High Resolution Computed Tomography, without a specific diagnosis of CTD. Three groups were identified according to patients' characteristics: IPAF, UCTD or idiopathic NSIP (iNSIP). Forty percent, 27% and 55% of patients showed diagnostic criteria for IPAF, UCTD and iNSIP, respectively. No significant differences in age, gender, smoking habit, pulmonary function tests and three-year survival rate were observed among study groups. IPAF patients with antisynthetase antibodies positivity, in comparison to IPAF without antisynthetase antibodies positivity, showed more frequently an acute onset (44% vs 9%, p<0.012). The presence of autoimmune features seems not to be associated with better outcomes in NSIP patients. IPAF criteria seem to be more representative than UCTD criteria in identifying patients with autoimmune features. Further studies are needed to verify if IPAF should include patients with positive antisynthetase serology.

Background: Screening for antinuclear antibodies by indirect immunofluorescence (ANA-IIF) is essential in the diagnostic workup of ANA-associated autoimmune rheumatic diseases (AARDs). However, also healthy individuals may test positive, making the interpretation challenging. Recent reports suggest that dense fine speckled 70 antibodies (anti-DFS70) may facilitate this challenge. Here, we investigate their clinical importance based on data from four Belgian laboratories (one primary, two secondary and one tertiary care)., Methods: At least one specific DFS70 assay (DFS70 IgG ELISA or lineblot [Euroimmun, full length antigen] and/or DFS70 IgG CLIA [Inova Diagnostics, truncated antigen]) was performed on four consecutive cohorts of homogeneous-like ANA-IIF samples (n=697). Co-occurrence with AARD-specific ANA and clinical information were documented in the anti-DFS70-positive samples., Results: Using a combination of solid phase techniques, we found between 7.6% and 26% anti-DFS70 in the different cohorts. Focusing on anti-DFS70 CLIA-positive samples without co-occurrence of AARD-specific ANA, we observed a trend towards lower frequency in tertiary (8% [p=0.0786]) and secondary care (12% [p=0.1275] and 6% [p<0.001]) compared to primary care (21%). Moreover, in this specific subpopulation, AARD was less frequent (0%-50% compared to 6%-77% in the total anti-DFS70-positive group)., Conclusions: Anti-DFS70 prevalence depends on the applied assay and care setting. Our data suggest that, for an ANA-IIF-positive patient, it is rather the absence of AARD-associated ANA and clinical symptoms that contribute to the exclusion of AARD than the presence of anti-DFS70. Nevertheless, isolated anti-DFS70 helps to clarify positive ANA-IIF results, especially if pretest probability for AARD is low.

CD38 is a transmembrane protein expressed in B lymphocytes, and is able to induce responses as proliferation, differentiation or apoptosis. Several reports propose that CD38 deficiency accelerates autoimmune processes in murine models of autoimmune diabetes, lymphoproliferation and rheumatoid arthritis. Other reports have shown elevated CD38 expression in B and T cells from patients with autoimmunity; however, the role of CD38 is still unclear in the development of autoimmunity. Recently, it has been characterized as CD1d hi CD5 + regulatory B cell subpopulation able to produce IL-10, and the loss of these cells exacerbates the autoimmunity in murine models. Here, we report that CD38 -/- mice exhibited elevated titres of ANAS, anti-dsDNA autoantibodies from 12 months of age and were higher by 16 months of age and mice presented kidney damage. Interestingly, there is a reduction in the survival of CD38 -/- mice compared to the WT. Furthermore, CD38 is highly expressed by CD1d high CD5 + regulatory B cells, and the agonistic anti-CD38 stimulus plus LPS was able to increase the percentage of this cell subset and its ability to induce IL-10 production. Together, these results suggest that CD38 could play a role in the control of autoimmune diseases through their expression on regulatory B cells., (© 2018 The Foundation for the Scandinavian Journal of Immunology.)

Dogs can spontaneously develop complex systemic autoimmune disorders, with similarities to human autoimmune disease. Autoantibodies directed at self-antigens are a key feature of these autoimmune diseases. Here we report the identification of interleukin enhancer-binding factors 2 and 3 (ILF2 and ILF3) as autoantigens in canine immune-mediated rheumatic disease. The ILF2 autoantibodies were discovered in a small, selected canine cohort through the use of human protein arrays; a method not previously described in dogs. Subsequently, ILF3 autoantibodies were also identified in the same cohort. The results were validated with an independent method in a larger cohort of dogs. ILF2 and ILF3 autoantibodies were found exclusively, and at a high frequency, in dogs that showed a speckled pattern of antinuclear antibodies on immunofluorescence. ILF2 and ILF3 autoantibodies were also found at low frequency in human patients with SLE and Sjögren's syndrome. These autoantibodies have the potential to be used as diagnostic biomarkers for canine, and possibly also human, autoimmune disease.

Objective: Although antinuclear autoantibody (ANA) staining of oral biopsy specimens is indicative of chronic ulcerative stomatitis, it is not known whether this staining is characteristic of other autoimmune diseases. Our study was undertaken to characterize the various in vivo ANA patterns detected in the oral mucosa by direct immunofluorescence to describe the associated hematoxylin and eosin findings, and determine whether patients with these findings had a coexisting systemic connective tissue disease., Study Design: This was a retrospective analysis of oral biopsy specimens submitted from 2013 to 2016., Results: In vivo ANA staining was present in 72 of the 2019 cases examined. Immunoglobulin G was the most common immunoreactant (71 of 72 cases), and speckled nuclear staining was the most frequent in vivo ANA pattern (52 of 72). In most cases, hematoxylin and eosin staining of biopsy specimens showed mucositis (24 of 34). Detailed clinical information was available for 10 patients, and all of them had an autoimmune disease., Conclusions: We found similar prevalence of ANA staining with direct immunofluorescence in oral epithelial biopsy specimens as reported for those of skin. In vivo ANA in the oral epithelium may indicate the presence of an immune-mediated disease. Patients who show ANA deposits in oral mucosal biopsy specimens should be investigated for systemic connective tissue disease as well as for chronic ulcerative stomatitis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Scurfy mice have a functional defect in regulatory T cells (Treg), which leads to lethal multi-organ inflammation. The missing Treg function results in uncontrolled autoimmune cellular and humoral inflammatory responses. We and others have previously shown that during the course of disease scurfy mice develop severe skin inflammation and autoantibodies including anti-nuclear autoantibodies (ANA)., Objective: Autoimmune skin inflammation and ANA are hallmarks for the diagnosis of autoimmune connective tissue diseases; therefore we analyzed scurfy mice for typical signs of these diseases., Methods: Indirect immunofluorescence was used to specify the ANA pattern in scurfy mice. Skin fibrosis was assessed by cutaneous collagen accumulation (Goldeners trichrome staining), collagen crosslinking/disorganization (Sirus red polarimetry) and quantitative PCR for fibrosis-related transcripts. The cellular components of the inflammatory infiltrates in scurfy skin were analyzed by flow cytometry and intracellular cytokine staining., Results: The majority of scurfy mice developed ANA with a predominant AC-5 pattern typical for mixed connective tissue disease, especially scleroderma. Scurfy mice showed higher skin collagen content compared to WT controls with a significant tendency in upregulation of TIMP-1. CD3 + CD4 + T cells in scurfy skin exhibited a strong Th2 deviation with a significant increase of IL-4, IL-5 and IL-13, and M2-polarized CD11b + MHCII + macrophages compared to WT mice., Conclusion: We show that Scurfy mice show a predominant AC-5 ANA pattern typical for mixed connective tissue disease as in scleroderma. The autoimmune inflammation in scurfy skin mainly consists of CD4 + T cells with Th2 differentiation and alternatively-activated (M2) macrophages as it is found in scleroderma with advanced fibrosis., (Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

Background: To identify the prognostic factors for survival in patients with interstitial pneumonia with autoimmune features (IPAF) who meet the serological domain of the IPAF criteria., Methods: We retrospectively analysed 99 IPAF patients who met the serological domain and were hospitalised at the Respiratory Medicine Unit of Kurashiki Central Hospital from 1999 to 2015. The high-resolution computed tomography findings were usual interstitial pneumonia (UIP; n = 1), non-specific interstitial pneumonia (NSIP; n = 63), NSIP with organizing pneumonia (OP) overlap (n = 15), and OP (n = 20). One patient who had radiological UIP pattern, and met the serological and clinical domains was excluded. The clinical characteristics, radiological findings, administered therapy, and prognosis of the remaining 98 IPAF patients who met the serological and morphological domains were analysed., Results: The median age of the 98 IPAF patients was 68 years, and 41 (41.8%) of them were men. Twelve (12.2%) of the 98 IPAF patients developed other characteristics and were diagnosed with connective tissue disease (CTD) later during the median follow-up of 4.5 years. Univariate Cox analysis revealed systemic sclerosis (SSc)-specific and SSc-associated antibodies (ANA nucleolar pattern, ANA centromere pattern, anti-ribonucleoprotein and anti-Scl-70) positive IPAF, radiological NSIP pattern, bronchoalveolar lavage fluid lymphocytes >15%, and age as significant prognostic factors for survival. Multivariate Cox analysis revealed radiological NSIP pattern (hazard ratio [HR], 4.48; 95% confidence interval [CI], 1.28-15.77, p = 0.02) and age (HR, 1.07; 95% CI, 1.02-1.11, p = 0.01) were significantly associated with worse survival., Conclusions: We confirmed that radiological NSIP pattern and age are poor prognostic factors for the survival of IPAF patients. This study suggested that the autoantibodies that are highly specific for certain connective tissue diseases might be less important for the prognosis of IPAF compared with the radiological-pathological patterns. The relatively high proportion of IPAF patients who developed CTD later suggests the importance of careful observation for evolution to CTD in IPAF.

Standardization and harmonization are complementary tools to achieve higher testing quality in laboratory medicine. Both are of great relevance and are strongly needed in autoimmune diagnostics, due to the impressive advance in basic research and technological development observed in this diagnostic field in recent years that has led to the introduction of many new tests and new analytical methods. It is, therefore, essential that this strong innovative thrust is translated into clinical practice in a coordinated way to avoid confusion and the risk of potentially harmful errors for the patient. However, while standardization of antibody assays is a very complex task, harmonization of procedures and behaviors is a more feasible target and should necessarily include all the phases of the total testing process-in the pre-analytical phase, appropriateness of test requests, harmonization of autoantibody terminology, and adoption of uniform nomenclature for laboratory tests; in the analytical phase, harmonization of measurements, and sharing of test profiles and diagnostic algorithms; and in the post-analytical phase, harmonization of data reporting, and criteria for interpreting immunoserological results, especially harmonization of units, reference intervals, decision limits, and definition and notification of critical values. We here provide and discuss some examples of harmonization initiatives related to anti-nuclear antibodies, TSH receptor, and anti-thyroid peroxidase antibodies and to antibodies associated with autoimmune hepatitis and with celiac disease. These initiatives could be the starting steps to achieve a wider consensus and a closer interaction among stakeholders in the path of autoimmune diagnostics harmonization to enhance clinical effectiveness and provide greater patient safety.

Objectives: Autoimmune congenital heart block (CHB) is associated with placental transcytosis of maternal autoantibodies directed against Ro/SS-A and La/SS-B. However, only about 2% of children born to mothers with the respective antibodies are affected, indicating that further risk factors exist, which are not yet fully understood. In this study, we investigated whether a maternal type I interferon (IFN) signature represents a risk factor for the development of CHB., Methods: Blood samples, clinical data and serological parameters from 9 women with CHB pregnancies, 14 pregnant women with antibodies against Ro/SS-A but without a CHB complication and another 30 healthy pregnant women as controls were studied. SIGLEC1 expression was measured by flow cytometry and was correlated to plasma IFN-α levels measured by ELISA, and IFN-γ-induced protein 10 (IP-10) levels measured by Bio-Plex technique., Results: Mothers of affected children had a significantly higher expression of SIGLEC1 (p=0.0034) and IFN-α (p=0.014), but not of IP-10 (p=0.14, all MWU) compared to mothers of unaffected children. SIGLEC1 and IFN-α expression were reduced by hydroxychloroquine and oral glucocorticoids., Conclusions: High expression of SIGLEC1 in pregnant women with autoantibodies against Ro/SS-A indicates an enhanced risk for CHB development, and these women may benefit especially from IFN-α directed therapy, for example with hydroxychloroquine., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Anecdotal case reports, amplified by mass media and internet-based opinion groups, have recently indicated vaccinations as possibly responsible for autoimmunity/lymphoproliferation development. Multiply vaccinated Italian military personnel (group 1, operating in Italy, group 2, operating in Lebanon) were followed-up for nine months to monitor possible post-vaccine autoimmunity/lymphoproliferation onset. No serious adverse event was noticed in both groups. Multivariate analysis of intergroup differences only showed a significant association between lymphocyte increase and tetanus/diphtheria vaccine administration. A significant post-vaccine decrease in autoantibody positivity was observed. Autoantibodies were also studied by microarray analysis of self-proteins in subjects exposed to ≥4 concurrent vaccinations, without observing significant difference among baseline and one and nine months post-vaccine. Moreover, HLA-A2 subjects have been analyzed for the possible CD8T-cell response to apoptotic self-epitopes, without observing significant difference between baseline and one month post-vaccine. Multiple vaccinations in young adults are safe and not associated to autoimmunity/lymphoproliferation onset during a nine-month-long follow-up., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Purpose of Review: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. Fever, rash and severe arthralgia are the hallmarks of chikungunya fever (CHIKF), the disease caused by this virus. The acute course of the disease usually lasts few weeks to months. Chronic, relapsing or persistent arthralgia and arthritis have been described mimicking rheumatoid arthritis (RA), requiring immunosuppressive drugs.The purpose of this review is to characterize both the chronic clinical course of CHIKF-associated arthritis and the immunological pathogenic mechanisms involved., Recent Findings: The effect of postepidemic chronic persistent rheumatic course on the functional status of affected individuals, affecting large populations, has been studied. One-third of affected individuals had persistent pain months to years postepidemic and the identified risk factors for functional disability were identified.Inflammatory biomarkers associated with disease severity of RA such as interleukin 6 (IL6), and relevant chemokines have been found to correlate with the severity of postepidemic chronic disease. There are conflicting reports on antinuclear antibodies (ANAs) as well as rheumatoid factor and anti-citrullinated peptide antibody (ACPA) sero-positivity during infections.According to a recent study, eight out of 10 infected individuals developed chronic persistent rheumatic course and met classification criteria for seronegative RA.In a flow cytology analyses, these eight patients, similar to a group of RA patients, had a greater percentage of activated and effector CD4 and CD8 T cells than healthy controls., Summary: Patients with CHKV infections may have a chronic persistent course of musculoskeletal disease, overlapping clinical and immunologic features with RA patients. In the appropriate setting and awareness, CHIKV infection should be considered when a patient is evaluated with a new symmetric polyarthritis.The question to be raised: Is it possible that in genetic prone individuals and in a particular environmental and infectious setting, such as CHIKF outbreak, an autoimmune disease will emerge?

Background Systemic lupus erythematosus (SLE) is an autoimmune disease with presence of autoantibodies and characteristic multi-organ involvement. Relatives of SLE patients have an increased risk of autoantibody production and autoimmune diseases. Methods In 2001, 226 first degree relatives (FDRs) of a population-based cohort of SLE patients were examined for the prevalence of autoantibodies and self-reported health complaints. In 2013, 143 FDRs were re-investigated and deceased's medical records were examined. Results Participants and non-participants were comparable regarding baseline characteristics, while deceased FDRs were older than participants, but with comparable ANA status. ANA status at baseline correlated to ANA status at follow-up. At follow-up, two FDRs reported SLE and 15 FDRs other autoimmune diseases. No observation at baseline alone could predict self-reported health. During follow-up 33 died at median age 76 years. Three deceased FDRs were diagnosed with an autoimmune disease. Conclusion The study showed that FDRs of SLE patients have an increased prevalence of ANA compared to healthy controls. The prevalence increased during follow-up, and ANA positive FDRs at baseline were prone to be ANA positive at follow-up. ANA positive FDRs had more self-reported autoimmune diseases, including SLE and rheumatoid arthritis, than reported from other population-based investigations.

Despite all the progress in the establishment of specific autoantibody assays, screening for antinuclear antibodies (ANA) by indirect immunofluorescence on HEp-2 cells for quality-oriented laboratory diagnosis of ANA associated rheumatic diseases (AARD) remains indispensable but is not without limitations. Recent data on the relevance of the dense fine speckled (DFS) pattern and anti-DFS70 antibodies disclosed novel possibilities to optimize the serological stepwise diagnostics of AARD. The DFS pattern on HEp-2 cells is well differentiated from the classic "homogeneous" ANA pattern associated with dsDNA antibodies. This is the most frequent pattern in high titer ANA-positive healthy persons. The most characteristic ANA specificity associated with DFS pattern is the anti-DFS70 antibody (synonym LEDGF antibody). The prevalence of anti-DFS70 antibodies in AARD patients is significantly lower compared with the prevalence in ANA-positive healthy persons. There is a negative association between anti-DFS70 antibodies and AARD, especially if no concomitant AARD-specific autoantibodies are found. Isolated anti-DFS70 antibodies are detectable in less than 1 % of AARD but are detectable in 2-22 % of healthy persons. In the presence of an isolated anti-DFS70 antibody, the posttest probability for AARD is reduced significantly. The significance of anti-DFS70 antibodies as a criterion that helps to exclude AARD is also confirmed by follow-up studies on anti-DFS70 antibodies of positive, healthy individuals, who did not develop any AARD during a 4 year observation period. Consequently, anti-DFS70 antibodies are valuable novel biomarkers for better interpretation of positive ANA in cases of negative AARD-associated autoantibodies and should be integrated in modified test algorithms to avoid unnecessary referrals and examinations of ANA-positive persons.

Background: Elevated levels of type I interferons (IFNs) are a characteristic feature of the systemic autoimmune rheumatic diseases (SARDs) and are thought to play an important pathogenic role. However, it is unknown whether these elevations are seen in anti-nuclear antibody-positive (ANA + ) individuals who lack sufficient criteria for a SARD diagnosis. We examined IFN-induced gene expression in asymptomatic ANA + individuals and patients with undifferentiated connective tissue disease (UCTD) to address this question., Methods: Healthy ANA - control subjects and ANA + titre (≥1:160 by immunofluorescence) participants meeting no criteria, meeting at least one criterion (UCTD) or meeting SARD classification criteria were recruited. Whole peripheral blood IFN-induced and BAFF gene expression were quantified using NanoString technology. The normalized levels of five IFN-induced genes were summed to produce an IFN5 score., Results: The mean IFN5 scores were increased in all ANA + participant subsets as compared with healthy control subjects. We found that 36.8% of asymptomatic ANA + and 50% of UCTD participants had IFN5 scores >2 SD above the mean for healthy control subjects. In all ANA + subsets, the IFN5 score correlated with the presence of anti-Ro/La antibodies. In the asymptomatic ANA + subset, this score also correlated with the ANA titre, whereas in the other ANA + subsets, it correlated with the number of different ANA specificities. Development of new SARD criteria was seen in individuals with normal and high IFN5 scores., Conclusions: An IFN signature is seen in a significant proportion of ANA + individuals and appears to be associated with ANA titre and type of autoantibodies, rather than with the presence or development of clinical SARD symptoms.