Anti-histone and anti-nucleosome rather than anti-dsDNA antibodies associate with IFN-induced biomarkers in Sudanese and Swedish Systemic Lupus Erythematosus patients.

OBJECTIVES: In SLE, anti-dsDNA can co-occur with autoantibodies against other chromatin components, like histones and nucleosomes. These antibodies induce type-1 interferon production, a hallmark of SLE. We measured antinuclear antibody (ANA) sub-specificities and investigated their associations to inflammatory biomarkers including interferon-regulated chemokines. METHODS: We included 93 Sudanese and 480 Swedish SLE patients and matched controls (N = 104 + 192). Autoantibodies targeting ANA-subspecificites: dsDNA, Sm, Sm/U1RNPcomplex, U1RNP, SSA/Ro52, SSA/Ro60, SSB/La, ribosomal P, PCNA and histones were quantified in all subjects, anti-nucleosome only in the Swedish patients, with a bead-based multiplex immunoassay. Levels of 72 plasma biomarkers were determined with Proximity Extension Assay technique or ELISA. RESULTS: Among Sudanese patients, the investigated antibodies significantly associated with 9/72 biomarkers. Anti-histone antibodies showed the strongest positive correlations with MCP-3 and S100A12 as well as with interferon I-inducible factors MCP-1 and CXCL10. Anti-dsDNA antibodies associated with CXCL10 and S100A12, but in multivariate analyses, unlike anti-histone, associations lost significance.Among Swedish patients, MCP-1, CXCL10, SA100A12 also demonstrated stronger associations to anti-histone and anti-nucleosome antibodies, compared with anti-dsDNA and other ANA sub-specificities. In multiple regression models, anti-histone/nucleosome retained the strongest associations. When excluding anti-histone or anti-nucleosome positive patients, the associations between MCP-1/CXCL10 and anti-dsDNA were lost. In contrast, when excluding anti-dsDNA positive patients, associations with anti-histone and anti-nucleosome remained significant. CONCLUSION: In two cohorts of different ethnical origin, autoantibodies targeting chromatin correlate stronger with IFN-induced inflammatory biomarkers than anti-dsDNA or other ANA sub-specificities. Our results suggest that anti-histone/nucleosome autoantibodies may be main drivers of type-1 interferon activity in SLE.

Hydroxychloroquine in lupus or rheumatoid arthritis pregnancy and risk of major congenital malformations: a population-based cohort study.

OBJECTIVES: To assess the infant risk of major congenital malformations (MCM) associated with first-trimester exposure to hydroxychloroquine (HCQ) among mothers with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA). METHODS: This population-based cohort study utilised Swedish nationwide registers and included all singleton births (2006-2021) among individuals with prevalent SLE or RA in Sweden. The exposure was filling ≥1 HCQ prescription during the first trimester. The outcome was infant MCM within one year of birth. Inverse probability of treatment weighting was applied to adjust for potential confounders (e.g. maternal smoking, body mass index, pregestational diabetes, and corticosteroids). Modified Poisson regression models with robust variance estimated risk ratios and 95% confidence intervals (RR 95%CI). RESULTS: We included 1,007 births (453 exposed) and 2,500 births (144 exposed) in the SLE and RA cohorts, respectively. The MCM risks in the SLE overall cohort, exposed, and unexposed groups were 3.6%, 3.7%, and 3.4%, respectively. The corresponding figures in the RA cohort were 4.4%, 5.6%, and 4.3%, respectively. The adjusted RRs (95%CI) were 1.29 (0.65-2.56) in the SLE cohort, 1.32 (0.56-3.13) in the RA cohort, and 1.30 (0.76-2.23) in the pooled analysis. The adjusted risk difference (exposed vs unexposed) was small (0.9% in SLE and 1.3% in RA). Sensitivity analyses examining different exposure and outcome windows yielded similar findings. CONCLUSIONS: First-trimester exposure to HCQ was not associated with a significantly increased risk of MCM. HCQ's benefits may outweigh the risks in managing SLE or RA during pregnancy.

Low CD4 + T cell count is related to specific anti-nuclear antibodies, IFNα protein positivity and disease activity in systemic lupus erythematosus pregnancy.

BACKGROUND: Lymphopenia, autoantibodies and activation of the type I interferon (IFN) system are common features in systemic lupus erythematosus (SLE). We speculate whether lymphocyte subset counts are affected by pregnancy and if they relate to autoantibody profiles and/or IFNα protein in SLE pregnancy. METHODS: Repeated blood samples were collected during pregnancy from 80 women with SLE and 51 healthy controls (HC). Late postpartum samples were obtained from 19 of the women with SLE. Counts of CD4 + and CD8 + T cells, B cells and NK cells were measured by flow cytometry. Positivity for anti-nuclear antibodies (ANA) fine specificities (double-stranded DNA [dsDNA], Smith [Sm], ribonucleoprotein [RNP], chromatin, Sjögren's syndrome antigen A [SSA] and B [SSB]) and anti-phospholipid antibodies (cardiolipin [CL] and ß2 glycoprotein I [ß2GPI]) was assessed with multiplexed bead assay. IFNα protein concentration was quantified with Single molecule array (Simoa) immune assay. Clinical data were retrieved from medical records. RESULTS: Women with SLE had lower counts of all lymphocyte subsets compared to HC throughout pregnancy, but counts did not differ during pregnancy compared to postpartum. Principal component analysis revealed that low lymphocyte subset counts differentially related to autoantibody profiles, cluster one (anti-dsDNA/anti-Sm/anti-RNP/anti-Sm/RNP/anti-chromatin), cluster two (anti-SSA/anti-SSB) and cluster three (anti-CL/anti-ß2GPI), IFNα protein levels and disease activity. CD4 + T cell counts were lower in women positive to all ANA fine specificities in cluster one compared to those who were negative, and B cell numbers were lower in women positive for anti-dsDNA and anti-Sm compared to negative women. Moreover, CD4 + T cell and B cell counts were lower in women with moderate/high compared to no/low disease activity, and CD4 + T cell count was lower in IFNα protein positive relative to negative women. Finally, CD4 + T cell count was unrelated to treatment. CONCLUSION: Lymphocyte subset counts are lower in SLE compared to healthy pregnancies, which seems to be a feature of the disease per se and not affected by pregnancy. Our results also indicate that low lymphocyte subset counts relate differentially to autoantibody profiles, IFNα protein levels and disease activity, which could be due to divergent disease pathways.

Regional european genetic ancestry predicts type I interferon level and risk of severe viral infection.

BACKGROUND: Viral infection outcomes vary widely between individuals, ranging from mild symptoms to severe organ failure and death, and it is clear that host genetic factors play a role in this variability. Type I interferon (IFN) is a critical anti-viral cytokine, and we have previously noted differences in type I IFN levels between world populations. METHODS: In this study, we investigate the interrelationship between regional European genetic ancestry, type I IFN levels, and severe viral infection outcomes. RESULTS: In cohorts of European ancestry lupus patients living in Europe, we noted higher IFN in the Northwestern populations as compared to Southeastern populations. In an independent cohort of European ancestry lupus patients from the United States with varying proportional regional European genetic admixture, we observed the same Northwest vs. Southeast European ancestry IFN gradient. We developed a model to predict type I IFN level based on regional European ancestry (AUC = 0.73, p = 6.1e-6). Examining large databases containing serious viral outcomes data, we found that lower predicted IFN in the corresponding European country was significantly correlated with increased viral infection fatality rate, including COVID-19, viral hepatitis, and HIV [Correlation coefficients: -0.79 (p = 4e-2), -0.94 (p = 6e-3), and -0.96 (p = 8e-2) respectively]. CONCLUSIONS: This association between predicted type I IFN level and viral outcome severity suggests a potential causal relationship, as greater intrinsic type I IFN is beneficial in host defense against viruses. Genetic testing could provide insight into individual and population level risk of fatality due to viruses prior to infection, across a wide range of viral pathogens.

A survey of ficolin-3 activity in Systemic Lupus Erythematosus reveals a link to hematological disease manifestations and autoantibody profile.

The complement system plays a central role in the pathogenesis of Systemic Lupus Erythematosus (SLE), but most studies have focused on the classical pathway. Ficolin-3 is the main initiator of the lectin pathway of complement in humans, but its role in systemic autoimmune disease has not been conclusively determined. Here, we combined biochemical and genetic approaches to assess the contribution of ficolin-3 to SLE risk and disease manifestations. Ficolin-3 activity was measured by a functional assay in serum or plasma samples from Swedish SLE patients (n = 786) and controls matched for age and sex (n = 566). Genetic variants in an extended 300 kb genomic region spanning the FCN3 locus were analyzed for their association with ficolin-3 activity and SLE manifestations in a Swedish multicenter cohort (n = 985). Patients with ficolin-3 activity in the highest tertile showed a strong enrichment in an SLE cluster defined by anti-Sm/DNA/nucleosome antibodies (OR 3.0, p < 0.001) and had increased rates of hematological disease (OR 1.4, p = 0.078) and lymphopenia (OR = 1.6, p = 0.039). Genetic variants associated with low ficolin-3 activity mapped to an extended haplotype in high linkage disequilibrium upstream of the FCN3 gene. Patients carrying the lead genetic variant associated with low ficolin-3 activity had a lower frequency of hematological disease (OR 0.67, p = 0.018) and lymphopenia (OR 0.63, p = 0.031) and fewer autoantibodies (p = 0.0019). Loss-of-function variants in the FCN3 gene were not associated with SLE, but four (0.5 %) SLE patients developed acquired ficolin-3 deficiency where ficolin-3 activity in serum was depleted following diagnosis of SLE. Taken together, our results provide genetic and biochemical evidence that implicate the lectin pathway in hematological SLE manifestations. We also identify lectin pathway activation through ficolin-3 as a factor that contributes to the autoantibody response in SLE.

EULAR recommendations for the management of systemic lupus erythematosus: 2023 update.

OBJECTIVES: To update the EULAR recommendations for the management of systemic lupus erythematosus (SLE) based on emerging new evidence. METHODS: An international Task Force formed the questions for the systematic literature reviews (January 2018-December 2022), followed by formulation and finalisation of the statements after a series of meetings. A predefined voting process was applied to each overarching principle and recommendation. Levels of evidence and strengths of recommendation were assigned, and participants finally provided their level of agreement with each item. RESULTS: The Task Force agreed on 5 overarching principles and 13 recommendations, concerning the use of hydroxychloroquine (HCQ), glucocorticoids (GC), immunosuppressive drugs (ISDs) (including methotrexate, mycophenolate, azathioprine, cyclophosphamide (CYC)), calcineurin inhibitors (CNIs, cyclosporine, tacrolimus, voclosporin) and biologics (belimumab, anifrolumab, rituximab). Advice is also provided on treatment strategies and targets of therapy, assessment of response, combination and sequential therapies, and tapering of therapy. HCQ is recommended for all patients with lupus at a target dose 5 mg/kg real body weight/day, considering the individual's risk for flares and retinal toxicity. GC are used as 'bridging therapy' during periods of disease activity; for maintenance treatment, they should be minimised to equal or less than 5 mg/day (prednisone equivalent) and, when possible, withdrawn. Prompt initiation of ISDs (methotrexate, azathioprine, mycophenolate) and/or biological agents (anifrolumab, belimumab) should be considered to control the disease and facilitate GC tapering/discontinuation. CYC and rituximab should be considered in organ-threatening and refractory disease, respectively. For active lupus nephritis, GC, mycophenolate or low-dose intravenous CYC are recommended as anchor drugs, and add-on therapy with belimumab or CNIs (voclosporin or tacrolimus) should be considered. Updated specific recommendations are also provided for cutaneous, neuropsychiatric and haematological disease, SLE-associated antiphospholipid syndrome, kidney protection, as well as preventative measures for infections, osteoporosis, cardiovascular disease. CONCLUSION: The updated recommendations provide consensus guidance on the management of SLE, combining evidence and expert opinion.

Systemic lupus erythematosus and damage: What has changed over the past 20 years?

The young age of onset and chronic/relapsing nature of systemic lupus erythematosus (SLE) make SLE patients prone to develop and accrue organ damage as a result of long-standing disease activity and side effects of treatment. There is a growing interest in objectifying damage and identifying its risk factors. Still, the lack of therapeutic alternatives has led to difficulties in avoiding immunosuppressives particularly corticosteroids, which have been implicated in a large spectrum of organ damage in SLE patients. Moreover, it continues to be very challenging to determine what actually causes damage in different organ-systems. Cardiovascular disease continues to be one of the leading types of damage in patients with SLE, reported as early as 1976. Since then, many researchers have focused on identifying SLE or treatment-related and traditional risk factors. The same considerations are valid for other conditions, such as the occurrence of metabolic syndrome, osteoporosis, avascular necrosis, susceptibility to infections, etc. On the other hand, diverse risk factors contribute to the development of chronic kidney disease (CKD) in SLE. Most evidence suggests that high initial levels of serum creatinine, hypocomplementemia, nephrotic range proteinuria, concomitant uncontrolled hypertension, Black and Hispanic ancestry, non-adherence to treatment, and biopsy findings such as diffuse proliferative lupus nephritis (LN), a high chronicity index, tubular atrophy, and tubulointerstitial inflammation are risk factors for progression to end stage renal disease (ESRD) in LN. While cardiovascular disease, CKD and infections are leading causes of mortality in patients with SLE, hospitalizations are caused mostly by SLE disease flares and infections. Cognitive impairment and mood disorders are common in SLE but continue to impose a challenge on how to measure, manage and decipher the underlying pathogenesis. Nevertheless, they have a great impact on SLE patients' health-related quality of life (HRQoL) and social functioning. Also, skin manifestations, such as alopecia and scaring, cataracts, and sicca symptoms result in a significant decrease in HRQoL. In light of recent developments in SLE treatment, we can expect to enter a period of new-age targeted therapies that will enable us to reduce disease activity and glucocorticoid usage further and positively alter the trajectory of damage development and accrual in SLE.

The expression of antibodies to Z-DNA in the blood of patients with systemic lupus erythematosus: Relationship to autoantibodies to B-DNA.

To explore the antibody response to Z-DNA, a DNA conformation with a zig-zag structure, blood of patients with systemic lupus erythematosus (SLE) and otherwise healthy individuals (NHS) were assayed by ELISA using brominated poly(dGdC), a synthetic Z-DNA antigen. These studies showed that SLE patients commonly express antibodies to Z-DNA; NHS also had binding in this assay. In SLE blood, levels of antibodies to Z-DNA were related to those to B-DNA using calf thymus DNA as a source of B-DNA; cross-reactivity was demonstrated by adsorption experiments using DNA cellulose. As shown by dissociation assays, antibody binding of SLE anti-Z-DNA is sensitive to the effects of ionic strength, suggesting electrostatic binding. Since Z-DNA structure can be found in bacterial DNA as well as bacterial biofilms, these findings suggest that, in SLE, anti-DNA antibody responses can result from stimulation by DNA of bacterial origin, with cross-reactivity leading to autoreactivity.

Urinary aberrations in systemic lupus erythematosus not always indicative of lupus nephritis: a cross-sectional cohort study.

INTRODUCTION: Kidney biopsy is the reference tool for diagnosing and guiding treatment strategies in inflammatory renal diseases, such as lupus nephritis (LN). We investigated the histopathological findings in first-time kidney biopsies from a large cohort of SLE patients. We focused on the occurrence and type of histopathological findings other than LN, and fulfillment of renal criteria in established SLE classification systems were analyzed. METHODS: We retrospectively included SLE patients (n = 139) who underwent a first kidney biopsy between 1995 and 2021, upon clinical suspicion of renal involvement. Based on histology, two groups were defined, LN and non-LN, for which clinical and laboratory features were compared. RESULTS: Findings consistent with LN according to ISN/RPS classification system were present in 123/139 patients (88.5%) and findings not consistent with LN were present in 16 /139 (11.5%). Non-LN patients were older at SLE diagnosis compared to LN patients (M, years 38.0 vs. 30.1, p=0.013) and had longer disease duration (M, years 11.9 vs 0.5) (p=0.027). Among non-LN patients 85.7% met the SLICC criteria item for renal SLE, seen in 94.7% in the LN group (ns). For the ACR/EULAR criteria, 66.7% of the non-LN group fulfilled the criteria compared to 74.8% in LN patients (ns). Proteinuria below the criteria cut-off level (< 0.5 g/24 h) was seen in 20% of patients with class III/IV LN. CONCLUSION: Our data confirm the importance of kidney biopsy for ruling out the presence of renal pathology other than LN. Patients with low-grade proteinuria may exhibit severe types of LN, which reinforces the need for early biopsies to detect LN. Key Points • Our findings show that histopathology changes other than lupus nephritis may occur in a significant number of patients with clinical and laboratory signs of novel kidney involvement. • Low-grade proteinuria does not exclude findings of active lupus nephritis that require the start of immunosuppressive therapy. • The study stresses the importance of performing kidney biopsies also in the presence of low-grade proteinuria or when signs of kidney function abnormalities occur. • This is crucial as early detection and prompt initiation of therapy may improve outcomes in lupus nephritis.

Low-density granulocytes are related to shorter pregnancy duration but not to interferon alpha protein blood levels in systemic lupus erythematosus.

BACKGROUND: An increased risk of pregnancy complications is seen in women with systemic lupus erythematosus (SLE), but the specific immunopathological drivers are still unclear. Hallmarks of SLE are granulocyte activation, type I interferon (IFN) overproduction, and autoantibodies. Here we examined whether low-density granulocytes (LDG) and granulocyte activation increase during pregnancy, and related the results to IFNα protein levels, autoantibody profile, and gestational age at birth. METHODS: Repeated blood samples were collected during pregnancy in trimesters one, two, and three from 69 women with SLE and 27 healthy pregnant women (HC). Nineteen of the SLE women were also sampled late postpartum. LDG proportions and granulocyte activation (CD62L shedding) were measured by flow cytometry. Plasma IFNα protein concentrations were quantified by single molecule array (Simoa) immune assay. Clinical data were obtained from medical records. RESULTS: Women with SLE had higher LDG proportions and increased IFNα protein levels compared to HC throughout pregnancy, but neither LDG fractions nor IFNα levels differed during pregnancy compared to postpartum in SLE. Granulocyte activation status was higher in SLE relative to HC pregnancies, and it was increased during pregnancy compared to after pregnancy in SLE. Higher LDG proportions in SLE were associated with antiphospholipid positivity but not to IFNα protein levels. Finally, higher LDG proportions in trimester three correlated independently with lower gestational age at birth in SLE. CONCLUSION: Our results suggest that SLE pregnancy results in increased peripheral granulocyte priming, and that higher LDG proportions late in pregnancy are related to shorter pregnancy duration but not to IFNα blood levels in SLE.

Urine Galectin-3 binding protein reflects nephritis activity in systemic lupus erythematosus.

BACKGROUND: Lupus nephritis (LN) is a major and severe organ involvement in systemic lupus erythematosus (SLE), whose diagnosis and treatment necessitate to perform kidney biopsy, which is an invasive procedure. Non-invasive urine biomarkers are an active area of investigation to support LN diagnosis and management. OBJECTIVE: To investigate the role of urinary galectin-3 binding protein (u-Gal-3BP) as a candidate biomarker of renal disease in biopsy proven LN. PATIENTS AND METHODS: Levels of u-Gal-3BP were investigated in a cross-sectional fashion by ELISA in 270 subjects: 86 LN patients, 63 active SLE patients with no kidney involvement, 73 SLE patients with inactive disease and 48 age and sex-matched population-based controls (PBC). Moreover, urine samples were analysed separately by ELISA for additional markers of kidney pathology: neutrophil gelatinase-associated lipocalin (NGAL), osteopontin (OPN), kidney injury molecule-1 (KIM-1) and galectin-3 (Gal-3). The concentrations of all studied molecules were normalized to urine creatinine levels. In 10 patients, post-treatment levels of the biomarkers were measured. RESULTS: Normalized u-Gal-3BP levels were higher in LN patients compared to the other groups (p < .0001). Comparing different LN classes, u-Gal-3BP levels were higher among patients with proliferative (class III/IV) and membranous (class V) as compared to mesangial (class II) forms (p = .04). In proliferative forms, u-Gal-3BP levels correlated with the activity index in renal biopsies (r = 0.42, p = .004). Moreover, in a subset of 10 patients with repeated kidney biopsy and urine sampling before and after induction treatment, a significant decrease of u-Gal-3BP was observed (p = .03). Among the other markers, KIM-1 was also able to discriminate LN from the other groups, while NGAL, OPN and Gal-3 could not in this cohort. CONCLUSION: Given its ability to discriminate LN patients from active non-renal and inactive SLE patients, the observed correlation with the activity index in renal biopsies, and its levels declining following treatment, u-Gal-3BP shows promise as a non-invasive urinary biomarker to help detecting and to monitor renal involvement in SLE patients and should be validated in larger cohorts.

Evaluating the Construct of Damage in Systemic Lupus Erythematosus.

OBJECTIVE: The Systemic Lupus International Collaborating Clinics (SLICC), American College of Rheumatology (ACR), and the Lupus Foundation of America are developing a revised systemic lupus erythematosus (SLE) damage index (the SLICC/ACR Damage Index [SDI]). Shifts in the concept of damage in SLE have occurred with new insights into disease manifestations, diagnostics, and therapy. We evaluated contemporary constructs in SLE damage to inform development of the revised SDI. METHODS: We conducted a 3-part qualitative study of international SLE experts. Facilitated small groups evaluated the construct underlying the concept of damage in SLE. A consensus meeting using nominal group technique was conducted to achieve agreement on aspects of the conceptual framework and scope of the revised damage index. The framework was finally reviewed and agreed upon by the entire group. RESULTS: Fifty participants from 13 countries were included. The 8 thematic clusters underlying the construct of SLE damage were purpose, items, weighting, reversibility, impact, time frame, attribution, and perspective. The revised SDI will be a discriminative index to measure morbidity in SLE, independent of activity or impact on the patient, and should be related to mortality. The SDI is primarily intended for research purposes and should take a life-course approach. Damage can occur before a diagnosis of SLE but should be attributable to SLE. Damage to an organ is irreversible, but the functional consequences on that organ may improve over time through physiological adaptation or treatment. CONCLUSION: We identified shifts in the paradigm of SLE damage and developed a unifying conceptual framework. These data form the groundwork for the next phases of SDI development.

Anti-phosphatidylserine/prothrombin antibodies and thrombosis associate positively with HLA-DRB1*13 and negatively with HLA-DRB1*03 in SLE.

OBJECTIVES: Emerging evidence demonstrates that aPS-PT associate with thrombotic events. Genetic predisposition, including HLA-DRB1 alleles, is known to contribute to the occurrence of conventional aPL [anti-ß2glycoprotein-I (anti-ß2GPI) and aCL]. We investigated associations between aPS-PT and HLA-DRB1* alleles and thrombosis in SLE. Conventional aPL were included for comparison. METHODS: We included 341 consecutive SLE patients, with information on general cardiovascular risk factors, including blood lipids, LA and thrombotic events. aPS/PT, anti-ß2GPI and aCL of IgA/G/M isotypes and LA were quantified. RESULTS: aPS/PT antibodies associated positively with HLA-DRB1*13 [odds ratio (OR) 2.7, P = 0.002], whereas anti-ß2GPI and aCL antibodies associated primarily with HLA-DRB1*04 (OR 2.5, P = 0.0005). These associations remained after adjustment for age, gender and other HLA-DRB1* alleles. HLA-DRB1*13, but not DRB1*04, remained as an independent risk factor for thrombosis and APS after adjustment for aPL and cardiovascular risk factors. The association between DRB1*13 and thrombosis was mediated by aPS-PT positivity. HLA-DRB1*03, on the other hand, associated negatively with thrombotic events as well as all aPL using both uni- and multivariate analyses. HLA-DRB1*03 had a thrombo-protective effect in aPL-positive patients. Additionally, HLA-DRB1*03 was associated with a favourable lipid profile regarding high-density lipoprotein and triglycerides. CONCLUSIONS: HLA-DRB1*13 confers risk for both aPS-PT and thrombotic events in lupus. The association between HLA-DRB1*13 and thrombosis is largely, but not totally, mediated through aPS-PT. HLA-DRB1*03 was negatively associated with aPL and positively with favourable lipid levels. Thus, HLA-DRB1*03 seems to identify a subgroup of SLE patients with reduced vascular risk.

Antiphospholipid antibodies in patients with calcific aortic valve stenosis.

OBJECTIVES: The antiphospholipid syndrome is defined by antiphospholipid antibodies (aPL) together with arterial and/or venous thromboembolism and/or obstetric morbidities. aPL are overrepresented in SLE and acute myocardial infarction, but it is unknown whether aPL are associated with calcific aortic valve stenosis (CAVS) in the general population. The prevalence of aPL and other SLE-associated autoantibodies and their impact on aortic valve transcriptomics were therefore determined. METHODS: A total of 233 tricuspid CAVS cases (median age 74, 69% male) and an age- and sex-matched control population were included. aPL were measured as anti-cardiolipin and anti-ß2Glycoprotein-I of IgG/M/A isotypes. Resilient, thickened and calcified aortic valve (AV) tissue derived from five aPL positive and five matched aPL negative CAVS patients undergoing surgical aortic valve replacement were analysed by microarrays. RESULTS: The prevalence of positivity for any aPL (IgG/M/A) in patients with CAVS was 6.4% (95% CI 3.6% - 10.4%: n = 233). aPL IgG was significantly more prevalent in CAVS cases vs controls (4.6% vs 0.6%, P = 0.04). AV tissue from aPL IgG/IgM-positive patients was negatively enriched in pathways related to interferon signalling. One hundred differentially expressed genes could predict local AV CAVS progression with supervised machine learning algorithms. CONCLUSIONS: aPL IgG was more common in CAVS patients compared with matched controls and aPL positivity was associated with altered AV transcriptomics related to local disease progression and interferon pathways. Further studies should aim to establish aPL as a possible risk marker and/or causal factor for CAVS and could offer new precision therapeutic targets.

Mer-tyrosine kinase: a novel susceptibility gene for SLE related end-stage renal disease.

OBJECTIVE: Lupus nephritis (LN) is a common and severe manifestation of SLE. The genetic risk for nephritis and progression to end-stage renal disease (ESRD) in patients with LN remains unclear. Herein, we aimed to identify novel genetic associations with LN, focusing on subphenotypes and ESRD. METHODS: We analysed genomic data on 958 patients with SLE (discovery cohort: LN=338) with targeted sequencing data from 1832 immunological pathway genes. We used an independent multiethnic cohort comprising 1226 patients with SLE (LN=603) as a replication dataset. Detailed functional annotation and functional epigenomic enrichment analyses were applied to predict functional effects of the candidate variants. RESULTS: A genetic variant (rs56097910) within the MERTK gene was associated with ESRD in both cohorts, meta-analysis OR=5.4 (2.8 to 10.6); p=1.0×10-6. We observed decreased methylation levels in peripheral blood cells from SLE patients with ESRD, compared with patients without renal SLE (p=2.7×10-4), at one CpG site (cg16333401) in close vicinity to the transcription start site of MERTK and located in a DNAse hypersensitivity region in T and B cells. Rs56097910 is linked to altered MERTK expression in kidney tissue in public eQTL databases. Two loci were replicated for association with proliferative LN: PRDM1 (rs6924535, pmeta=1.6×10-5, OR=0.58) and APOA1BP (NAXE) (rs942960, pmeta=1.2×10-5, OR=2.64). CONCLUSION: We identified a novel genetic risk locus, MERTK, associated with SLE-ESRD using the data from two large SLE cohorts. Through DNA methylation analysis and functional annotation, we showed that the risk could be mediated through regulation of gene expression. Our results suggest that variants in the MERTK gene are important for the risk of developing SLE-ESRD and suggest a role for PRDM1 and APOA1BP in proliferative LN.

Interleukin (IL) 16: a candidate urinary biomarker for proliferative lupus nephritis.

OBJECTIVE: Lupus nephritis (LN) is a severe manifestation of systemic lupus erythematosus (SLE). The pathogenesis is incompletely understood and diagnostic biomarkers are scarce. We investigated interleukin (IL) 16 as a potential biomarker for LN in a well-characterised cohort of patients with SLE. METHODS: We measured urinary (u-) and plasma (p-) levels of IL-16 in predefined patient groups using ELISA: LN (n=84), active non-renal SLE (n=63), inactive non-renal SLE (n=73) and matched population controls (n=48). The LN group included patients with recent biopsy-confirmed proliferative (PLN, n=47), mesangioproliferative (MES, n=11) and membranous (MLN, n=26) LN. Renal expression of IL-16 was investigated by immunohistochemistry. Associations between IL-16 measurements and clinical parameters and the diagnostic value for LN were explored. RESULTS: p-IL-16 was detected in all investigated cases and high p-IL-16 levels were observed in patients with active SLE. u-IL-16 was detected (dt-u-IL-16) in 47.6% of patients with LN, while only up to 17.8% had dt-u-IL-16 in other groups. In the LN group, 68% of patients with PLN had dt-u-IL-16, while the proportions in the MLN and MES groups were lower (11.5% and 45.5%, respectively). The highest u-IL-16 levels were detected in the PLN group. In the regression model, u-IL-16 levels differentiated PLN from other LN patient subgroups (area under the curve 0.775-0.896, p<0.0001). dt-u-IL-16 had superior specificity but slightly lower sensitivity than elevated anti-double-stranded DNA and low complement C3 or C4 in diagnosing PLN. A high proportion of LN kidney infiltrating cells expressed IL-16. CONCLUSIONS: We demonstrate that detectable u-IL-16 can differentiate patients with PLN from those with less severe LN subtypes and active non-renal SLE. Our findings suggest that u-IL-16 could be used as a screening tool at suspicion of severe LN. Furthermore, the high IL-16 levels in plasma, urine and kidney tissue imply that IL-16 could be explored as a therapeutic target in SLE.

Long-term prognosis after a first myocardial infarction: eight years follow up of the case-control study PAROKRANK.

Objective. To explore long-term cardiovascular outcomes and mortality in patients after a first myocardial infarction (MI) compared with matched controls in a contemporary setting. Methods. During 2010-2014 the Swedish study PAROKRANK recruited 805 patients <75 years with a first MI and 805 age-, gender-, and area-matched controls. All study participants were followed until 31 December 2018, through linkage with the National Patient Registry and the Cause of Death Registry. The primary endpoint was the first of a composite of all-cause death, non-fatal MI, non-fatal stroke, and heart failure hospitalization. Event rates in cases and controls were calculated using a Cox regression model, subsequently adjusted for baseline smoking, education level, and marital status. Kaplan-Meier curves were computed and compared by log-rank test. Results. A total of 804 patients and 800 controls (mean age 62 years; women 19%) were followed for a mean of 6.2 (0.2-8.5) years. The total number of primary events was 211. Patients had a higher event rate than controls (log-rank test p < .0001). Adjusted hazard ratio (HR) for the primary outcome was 2.04 (95% CI 1.52-2.73). Mortality did not differ between patients (n = 38; 4.7%) and controls (n = 35; 4.4%). A total of 82.5% patients and 91.3% controls were event-free during the follow up. Conclusions. In this long-term follow up of a contemporary, case-control study, the risk for cardiovascular events was higher in patients with a previous first MI compared with their matched controls, while mortality did not differ. The access to high quality of care and cardiac rehabilitation might partly explain the low rates of adverse outcomes.

Strong Association of Combined Genetic Deficiencies in the Classical Complement Pathway With Risk of Systemic Lupus Erythematosus and Primary Sjögren's Syndrome.

OBJECTIVE: Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjögren's syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. METHODS: The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. RESULTS: Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 × 10-9 ) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. CONCLUSION: We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS.

Complement C4 Copy Number Variation is Linked to SSA/Ro and SSB/La Autoantibodies in Systemic Inflammatory Autoimmune Diseases.

OBJECTIVE: Copy number variation of the C4 complement components, C4A and C4B, has been associated with systemic inflammatory autoimmune diseases. This study was undertaken to investigate whether C4 copy number variation is connected to the autoimmune repertoire in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (SS), or myositis. METHODS: Using targeted DNA sequencing, we determined the copy number and genetic variants of C4 in 2,290 well-characterized Scandinavian patients with SLE, primary SS, or myositis and 1,251 healthy controls. RESULTS: A prominent relationship was observed between C4A copy number and the presence of SSA/SSB autoantibodies, which was shared between the 3 diseases. The strongest association was detected in patients with autoantibodies against both SSA and SSB and 0 C4A copies when compared to healthy controls (odds ratio [OR] 18.0 [95% confidence interval (95% CI) 10.2-33.3]), whereas a weaker association was seen in patients without SSA/SSB autoantibodies (OR 3.1 [95% CI 1.7-5.5]). The copy number of C4 correlated positively with C4 plasma levels. Further, a common loss-of-function variant in C4A leading to reduced plasma C4 was more prevalent in SLE patients with a low copy number of C4A. Functionally, we showed that absence of C4A reduced the individuals' capacity to deposit C4b on immune complexes. CONCLUSION: We show that a low C4A copy number is more strongly associated with the autoantibody repertoire than with the clinically defined disease entities. These findings may have implications for understanding the etiopathogenetic mechanisms of systemic inflammatory autoimmune diseases and for patient stratification when taking the genetic profile into account.

Inflammatory markers in saliva and urine reflect disease activity in patients with systemic lupus erythematosus.

BACKGROUND: Laboratory tests of blood and sometimes urine are used to diagnose and to monitor disease activity (DA) in SLE. Clinical practice would be simplified if non-invasive urine and salivary tests could be introduced as alternatives to blood samples. We therefore explored the levels of innate immunity-related biomarkers in matched serum, urine and saliva samples from patients with SLE. METHODS: A total of 84 patients with SLE selected to represent high and low general DA, and 21 controls were included. All participants underwent a thorough clinical examination. General DA and renal DA were measured. The levels of colony-stimulating factor (CSF)-1, interleukin (IL)-34, tumour necrosis factor (TNF)-α, interferon-γ-induced protein (IP)-10, monocyte chemoattractant protein (MCP)-1, calprotectin, macrophage inflammatory protein (MIP)-1α and MIP-1ß were analysed by immunoassays and related to DA. RESULTS: CSF-1, TNF-α, IP-10 and MCP-1 in saliva, serum and urine, as well as calprotectin in saliva and urine were increased in patients with SLE as compared with controls (p<0.05). TNF-α, IP-10 and MCP-1 in saliva, serum and urine, and CSF-1 in saliva and serum distinguished patients with SLE from controls (area under the curve >0.659; p<0.05 for all). CSF-1 in serum and urine, and calprotectin in saliva and urine, as well as TNF- α, IP-10 and MCP-1 in urine correlated positively with measures of general DA (p<0.05). Patients with SLE with active renal disease presented elevated levels of TNF-α, IP-10 and MCP-1 in urine and CSF-1 and IP-10 in serum as compared with patients with SLE with non-active renal disease. CONCLUSIONS: Our investigation demonstrates that saliva is a novel alternative body fluid, with potential for surveillance of general DA in patients with SLE, but urine is more informative in patients with SLE with predominantly renal DA.