An AI-based imaging flow cytometry approach to study erythrophagocytosis.

Erythrophagocytosis is a process consisting of recognition, engulfment and digestion by phagocytes of antibody-coated or damaged erythrocytes. Understanding the dynamics that are behind erythrophagocytosis is fundamental to comprehend this cellular process under specific circumstances. Several techniques have been used to study phagocytosis. Among these, an interesting approach is the use of Imaging Flow Cytometry (IFC) to distinguish internalization and binding of cells or particles. However, this method requires laborious analysis. Here, we introduce a novel approach to analyze the phagocytosis process by combining Artificial Intelligence (AI) with IFC. Our study demonstrates that this approach is highly suitable to study erythrophagocytosis, categorizing internalized, bound and non-bound erythrocytes. Validation experiments showed that our pipeline performs with high accuracy and reproducibility.

Return-to-work following shoulder arthroplasty in adults with osteoarthritis.

BACKGROUND: Anatomic total shoulder arthroplasty (aTSA) and hemiarthroplasty (HA) have demonstrated significant improvement in shoulder function and pain relief. Work-related outcomes have become increasingly important, while the current literature lacks evidence related to return-to-work (RTW) and which factors might have an influence on it. AIMS: This study aimed to assess RTW in patients who have received aTSA or HA at a minimum of 1-year follow-up after surgery, and secondary to evaluate possible prognostic factors associated with RTW. METHODS: We performed a retrospective query in employed patients diagnosed with primary osteoarthritis of the shoulder, who received either an aTSA or HA between February 2006 and February 2021. Preoperative and post-operative work and sports participation were assessed. RESULTS: Forty-four patients participated in this study (98% compliance), of which 40 patients (91%) were able to RTW at a median time of two (interquartile range: 2-4) months post-operatively. Patients with a medium-/high-demand occupation demonstrated RTW at a significantly lower rate (79%) than those with light-demand occupations (100%; P = 0.03). There was a statistically significant association between return to full employment and patients' expectation to fully return, absence of preoperative work adjustments and preoperative sick leave (odds ratio: 16.9 [3.1-93.5]; 18.3 [2.1-160.4]; 0.1 [0.0-0.6]). CONCLUSIONS: aTSA and HA facilitate excellent RTW rates. Patients with a medium-/high-demand occupation return at a significantly lower rate. The ability to RTW seems to be multifactorial and the results found might not be attributed to shoulder arthroplasty alone.

Hemolysis in the spleen drives erythrocyte turnover.

Red pulp macrophages (RPMs) of the spleen mediate turnover of billions of senescent erythrocytes per day. However, the molecular mechanisms involved in sequestration of senescent erythrocytes, their recognition, and their subsequent degradation by RPMs remain unclear. In this study, we provide evidence that the splenic environment is of substantial importance in facilitating erythrocyte turnover through induction of hemolysis. Upon isolating human spleen RPMs, we noted a substantial lack of macrophages that were in the process of phagocytosing intact erythrocytes. Detailed characterization of erythrocyte and macrophage subpopulations from human spleen tissue led to the identification of erythrocytes that are devoid of hemoglobin, so-called erythrocyte ghosts. By using in vivo imaging and transfusion experiments, we further confirmed that senescent erythrocytes that are retained in the spleen are subject to hemolysis. In addition, we showed that erythrocyte adhesion molecules, which are specifically activated on aged erythrocytes, cause senescent erythrocytes to interact with extracellular matrix proteins that are exposed within the splenic architecture. Such adhesion molecule-driven retention of senescent erythrocytes under low shear conditions was found to result in steady shrinkage of the cell and ultimately resulted in hemolysis. In contrast to intact senescent erythrocytes, the remnant erythrocyte ghost shells were prone to recognition and breakdown by RPMs. These data identify hemolysis as a key event in the turnover of senescent erythrocytes, which alters our current understanding of how erythrocyte degradation is regulated.

Cost and impact of early diagnosis in primary immunodeficiency disease: A literature review.

BACKGROUND: New, innovative, costly diagnostic methods for patients with primary immunodeficiencies (PID) demand upfront insight into their potential cost savings and added value for individual patients. As such, high quality, comparable economic evaluations are of utmost importance to enable informed decisions. The objective of this review was therefore to create an extensive overview of current costing studies and potential cost savings of early diagnosis in primary immunodeficiency disease. METHODS: A literature search in PubMed was conducted and studies involving any form of costing study in the field of PIDs were included. Of the included studies, study characteristics, cost parameters and benefits of early diagnosis were extracted and outlined in separate tables. RESULTS: Twenty two studies met the inclusion criteria and were included in the review. The papers were categorized according to their subject: neonatal screening for severe combined immunodeficiency (SCID), Ig replacement therapies and studies reporting on costs of general or specific PIDs. Within and between these groups variability in reported costing characteristics was observed. In studies that reported cost savings pre- and post-diagnosis, cost savings ranged from 6500 to 108,463 USD of total costs per patient. CONCLUSION: This literature review shows that, regardless of what aspect of PIDs has been studied, in nearly all cases early diagnosis reduces health care consumption and leads to better health outcomes for patients with PIDs. We found considerable variability in costing characteristics of economic evaluations of PID patients, which hampers the comparability of outcomes. More effort is needed to create uniformity and define cost parameters in economic evaluations in the field of PIDs, facilitating further prospective research to extensively assess the benefits of early diagnosis.

Coronary artery assessment in Kawasaki disease with dual-source CT angiography to uncover vascular pathology.

BACKGROUND: Kawasaki disease (KD) is a vasculitis with formation of coronary artery aneurysms (CAAs) that can lead to myocardial ischemia. Echocardiography is the primary imaging modality for the coronary arteries despite limited visualization. Coronary angiography (CAG) is the gold standard yet invasive with high-radiation exposure. To date however, state-of-the-art CT scanners enable high-quality low-dose coronary computed tomographic angiography (cCTA) imaging. The aim of our study in KD is to report (i) the diagnostic yield of cCTA compared to echocardiography, and (ii) the radiation dose. METHODS AND RESULTS: We collected data of KD patients who underwent cCTA. cCTA findings were compared with echocardiography results. In 70 KD patients (median age 15.1 years [0.5-59.5 years]; 78% male; 38% giant CAA), the cCTA identified 61 CAAs, of which 34 (56%, with a Z score > 3, in 22 patients) were not detected by echocardiography. In addition, the left circumflex (aneurysmatic in 6 patients) was always visible upon cCTA and not detected upon echocardiography. Calcifications, plaques, and/or thrombi were visualized by cCTA in 25 coronary arteries (15 patients). Calcifications were seen as early as 2.7 years after onset of disease. In 5 patients, the cCTA findings resulted in an immediate change of treatment. The median effective dose (ED) in millisievert differed significantly (p < 0.01) between third-generation dual-source and other CT scanners (1.5 [0.3-9.4] (n = 56) vs 3.8 [1.7-20.0] (n = 14)). CONCLUSIONS: The diagnostic yield of third-generation dual-source cCTA combined with reduced radiation exposure makes cCTA a favorable diagnostic modality to complete the diagnosis and long-term treatment indications for KD. KEY POINTS: • cCTA is a favorable diagnostic modality to complete the diagnosis and long-term treatment indications for Kawasaki disease. • Kawasaki disease patients with proven coronary artery involvement on echocardiography require additional imaging.

DynOVis: a web tool to study dynamic perturbations for capturing dose-over-time effects in biological networks.

BACKGROUND: The development of high throughput sequencing techniques provides us with the possibilities to obtain large data sets, which capture the effect of dynamic perturbations on cellular processes. However, because of the dynamic nature of these processes, the analysis of the results is challenging. Therefore, there is a great need for bioinformatics tools that address this problem. RESULTS: Here we present DynOVis, a network visualization tool that can capture dynamic dose-over-time effects in biological networks. DynOVis is an integrated work frame of R packages and JavaScript libraries and offers a force-directed graph network style, involving multiple network analysis methods such as degree threshold, but more importantly, it allows for node expression animations as well as a frame-by-frame view of the dynamic exposure. Valuable biological information can be highlighted on the nodes in the network, by the integration of various databases within DynOVis. This information includes pathway-to-gene associations from ConsensusPathDB, disease-to-gene associations from the Comparative Toxicogenomics databases, as well as Entrez gene ID, gene symbol, gene synonyms and gene type from the NCBI database. CONCLUSIONS: DynOVis could be a useful tool to analyse biological networks which have a dynamic nature. It can visualize the dynamic perturbations in biological networks and allows the user to investigate the changes over time. The integrated data from various online databases makes it easy to identify the biological relevance of nodes in the network. With DynOVis we offer a service that is easy to use and does not require any bioinformatics skills to visualize a network.

Regulatory T cell features in chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (Tregs ) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in Tregs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated Tregs from gp91phox -, p47phox - and p40phox -deficient CGD patients separately. Results show that Treg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91phox -deficiency effector Treg (eTreg ) numbers are decreased. Expression of Treg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in Treg functionality or activation state. No correlation was seen between eTreg numbers and patients' clinical phenotype. To conclude, the only difference between Tregs from CGD patients and healthy controls is a decrease in circulating eTregs in gp91phox -deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived Tregs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for Tregs in CGD-related autoinflammation/autoimmunity.

Characterization of buffy coat-derived granulocytes for clinical use: a comparison with granulocyte colony-stimulating factor/dexamethasone-pretreated donor-derived products.

BACKGROUND AND OBJECTIVES: Buffy coat-derived granulocytes have been described as an alternative to the apheresis product from donors pretreated with dexamethasone and granulocyte colony-stimulating factor (G-CSF). The latter is - dependent on the local and national settings - obtained following a demanding and time-consuming procedure, which is undesirable in critically ill septic patients. In contrast, buffy coat-derived products have a large volume and are often heavily contaminated with red cells and platelets. We developed a new pooled buffy coat-derived product with high purity and small volume, and performed a comprehensive functional characterization of these granulocytes. MATERIALS AND METHODS: We pooled ten buffy coats following the production of platelet concentrates. Saline 0·9% was added to decrease the viscosity and the product was split into plasma, red cells and a 'super' buffy coat. Functional data of the granulocytes were compared to those obtained with granulocytes from healthy controls and G-CSF/dexamethasone-pretreated donors. RESULTS: Buffy coat-derived granulocytes showed adhesion, chemotaxis, reactive oxygen species production, degranulation, NETosis and in vitro killing of Staphylococcus aureus, Escherichia coli and Aspergillus species comparable to control and G-CSF/dexamethasone-derived granulocytes. Candida killing was superior compared to G-CSF/dexamethasone-derived granulocytes. Immunophenotyping was normal; especially no signs of activation in the buffy coat-derived granulocytes were seen. Viability was reduced. Buffy coats are readily available in the regular blood production process and would take away the concerns around the apheresis product. CONCLUSION: The product described appears a promising alternative for transfusion purposes.

Dissecting Kawasaki disease: a state-of-the-art review.

Kawasaki disease (KD) is a pediatric vasculitis with coronary artery aneurysms (CAA) as its main complication. The diagnosis is based on the presence of persistent fever and clinical features including exanthema, lymphadenopathy, conjunctival injection, and changes to the mucosae and extremities. Although the etiology remains unknown, the current consensus is that it is likely caused by an (infectious) trigger initiating an abnormal immune response in genetically predisposed children. Treatment consists of high dose intravenous immunoglobulin (IVIG) and is directed at preventing the development of CAA. Unfortunately, 10-20% of all patients fail to respond to IVIG and these children need additional anti-inflammatory treatment. Coronary artery lesions are diagnosed by echocardiography in the acute and subacute phases. Both absolute arterial diameters and z-scores, adjusted for height and weight, are used as criteria for CAA. Close monitoring of CAA is important as ischemic symptoms or myocardial infarction due to thrombosis or stenosis can occur. These complications are most likely to arise in the largest, so-called giant CAA. Apart from the presence of CAA, it is unclear whether KD causes an increased cardiovascular risk due to the vasculitis itself. CONCLUSION: Many aspects of KD remain unknown, although there is growing knowledge on the etiology, treatment, and development and classification of CAA. Since children with previous KD are entering adulthood, long-term follow-up is increasingly important. What is known: • Kawasaki disease (KD) is a pediatric vasculitis with coronary artery damage as its main complication. • Although KD approaches its 50th birthday since its first description, many aspects of the disease remain poorly understood. What is new: • In recent years, multiple genetic candidate pathways involved in KD have been identified, with recently promising information about the ITPKC pathway. • As increasing numbers of KD patients are reaching adulthood, increasing information is available about the long-term consequences of coronary artery damage and broader cardiovascular risk.

Brief pain re-assessment provided more accurate prognosis than baseline information for low-back or shoulder pain.

BACKGROUND: Research investigating prognosis in musculoskeletal pain conditions has only been moderately successful in predicting which patients are unlikely to recover. Clinical decision making could potentially be improved by combining information taken at baseline and re-consultation. METHODS: Data from four prospective clinical cohorts of adults presenting to UK and Dutch primary care with low-back or shoulder pain was analysed, assessing long-term disability at 6 or 12 months and including baseline and 4-6 week assessments of pain. Baseline versus short-term assessments of pain, and previously validated multivariable prediction models versus repeat assessment, were compared to assess predictive performance of long-term disability outcome. A hypothetical clinical scenario was explored which made efficient use of both baseline and repeated assessment to identify patients likely to have a poor prognosis and decide on further treatment. RESULTS: Short-term repeat assessment of pain was better than short-term change or baseline score at predicting long-term disability improvement across all cohorts. Short-term repeat assessment of pain was only slightly more predictive of long-term recovery (c-statistics 0.78, 95% CI 0.74 to 0.83 and 0.75, 95% CI 0.69 to 0.82) than a multivariable baseline prognostic model in the two cohorts presenting such a model (c-statistics 0.71, 95% CI 0.67 to 0.76 and 0.72, 95% CI 0.66 to 0.78). Combining optimal prediction at baseline using a multivariable prognostic model with short-term repeat assessment of pain in those with uncertain prognosis in a hypothetical clinical scenario resulted in reduction in the number of patients with an uncertain probability of recovery, thereby reducing the instances where patients may be inappropriately referred or reassured. CONCLUSIONS: Incorporating short-term repeat assessment of pain into prognostic models could potentially optimise the clinical usefulness of prognostic information.

Regression and Complications of z-score-Based Giant Aneurysms in a Dutch Cohort of Kawasaki Disease Patients.

Kawasaki disease (KD) is a pediatric vasculitis. Its main complication is the development of coronary artery aneurysms (CAA), with giant CAA at the end of the spectrum. We evaluated regression and event-free rates in a non-Asian cohort of patients with giant CAA using the current z-scores adjusted for body surface area instead of absolute diameters. KD patients with giant CAA (z-score ≥10) visiting our outpatient clinic between January 1999 and September 2015 were included. Patient characteristics and clinical details were extracted from medical records. Regression was defined as all coronary arteries having a z-score of ≤3. A major adverse event was defined as cardiac death, myocardial infarction, cardiogenic shock, or any coronary intervention. Regression-free and event-free rates were calculated using the Kaplan-Meier method. We included 52 patients with giant CAA of which 45 had been monitored since the acute phase. The 1-, 2-, and 5-year regression-free rates were 0.86, 0.78, and 0.65, respectively. The 5-year, 10-year, and 15-year event-free rates were 0.79, 0.75, and 0.65, respectively. Four children, whose CAA would not have been classified as 'giant' based on absolute diameters instead of z-scores, had experienced an event during follow-up. CONCLUSION: We found a high percentage of children in whom the lumen of giant CAA completely normalized. Four children not classified as 'giant' based on absolute diameters with z-scores of ≥10 experienced a cardiac event. Hence, the use of z-scores seems to be justified.

Novel insights in the genomic organization and hotspots of recombination in the human KIR locus through analysis of intergenic regions.

The Killer Immunoglobulin-like Receptor (KIR) proteins constitute a family of highly homologous surface receptors involved in the regulation of the innate cytotoxicity of natural killer (NK) cells. Within the human genome, 17 KIR genes are present, many of which show large variation across the population owing to the high number of allelic variants and copy number variation (CNV). KIR genotyping and CNV determination were used to map the KIR locus in a large cohort of >400 Caucasian individuals. Gene order and structure was determined by sequence-specific polymerase chain reaction of the intergenic regions. In this way, we could show that KIR3DL1 and KIR2DS4 gene variants are linked and that--contrary to current views--the gene KIR2DS5 is only present in the telomeric half of the KIR locus. Our study revealed novel insights in the highly organized distribution of KIR genes. Novel recombination hotspots were identified that contribute to the diversity of KIR gene distribution in the Caucasian population. Next-generation sequencing of the KIR intergenic regions allowed for a detailed single-nucleotide polymorphism analysis, which demonstrated several gene-specific as well as haplotype-specific nucleotides for a more accurate genotyping of this notoriously complex gene cluster.

Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

Primary immunodeficiencies in the Netherlands: national patient data demonstrate the increased risk of malignancy.

PURPOSE: To analyze the data of the national registry of all Dutch primary immune deficiency (PID) patients, according to the European Society for Immunodeficiencies (ESID) definitions. RESULTS: In the Netherlands, 745 patients had been registered between 2009 and 2012. An overall prevalence of 4.0 per 100,000 inhabitants was calculated. The most prevalent PID was 'predominantly antibody disorder (PAD)' (60.4%). In total, 118 transplantations were reported, mostly hematopoietic stem cell transplantations (HSCT). Almost 10% of the PID patients suffered from a malignancy, in particular 'lymphoma' and 'skin cancer'. Compared to the general Dutch population, the relative risk of developing any malignancy was 2.3-fold increased, with a >10-fold increase for some solid tumors (thymus, endocrine organs) and hematological disease (lymphoma, leukemia), varying per disease category. CONCLUSIONS: The incidence rate and characteristics of PID in the Netherlands are similar to those in other European countries. Compared to the general population, PID patients carry an increased risk to develop a malignancy.

A missense mutation underlies defective SOCS4 function in a family with autoimmunity.

OBJECTIVE: The aim of this study was to determine the genetic and immunological defects underlying familial manifestations of an autoimmune disorder. METHODS: Whole-exome sequencing was performed on the index patient with various manifestations of autoimmunity, including hypothyroidism, vitiligo and alopecia. Peripheral blood mononuclear cells and DNA of family members were used for functional and genetic testing of the candidate variants obtained by Sanger sequencing. RESULTS: Exome sequencing identified 233 rare, coding and nonsynonymous variants in the index patient; five were highly conserved and affect genes that have a possible role in autoimmunity. Only a heterozygous missense mutation in the suppressor of cytokine signalling 4 gene (SOCS4) cosegregated with the autoimmune disorder in the family. SOCS4 is a known inhibitor of epidermal growth factor (EGF) receptor signalling, and functional studies demonstrated specific upregulation of EGF-dependent immune stimulation in affected family members. CONCLUSION: We present a family with an autoimmune disorder, probably resulting from dysregulated immune responses due to mutations in SOCS4.

Therapies in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a genetically determined disorder, affecting most particularly the brain and the skin, characterized by the inappropriate induction of a type I interferon-mediated immune response. In most, but not all, cases the condition is severe, with a high associated morbidity and mortality. A number of important recent advances have helped to elucidate the biology of the AGS-related proteins, thus providing considerable insight into disease pathology. In this study, we outline the clinical phenotype of AGS, paying particular attention to factors relevant to therapeutic intervention. We then discuss the pathogenesis of AGS from a molecular and cell biology perspective. Finally, we suggest possible treatment strategies in light of these emerging insights.

The value of DNA storage and pedigree analysis in rare diseases: a 17-year-old boy with X-linked lymphoproliferative disease (XLP) caused by a de novo SH2D1A mutation.

UNLABELLED: We present a case of a fatal Epstein-Barr infection in a 17-year-old male patient suspected to be caused by X-linked lymphoproliferative disease. At the time of hospitalization, DNA diagnostics was not available. The suspected diagnosis was confirmed several years later when a SH2D1A missense mutation was identified in stored patient DNA. Extended pedigree analysis showed that this mutation occurred de novo in his mother. In addition, we provide a summary of the currently listed SH2D1A mutations. CONCLUSION: This case report underlines the importance of DNA storage, pedigree analysis, and multidisciplinary care in patients with rare diseases and their families.

Differential expression pattern of Bcl-2 family members in B and T cells in systemic lupus erythematosus and rheumatoid arthritis.

OBJECTIVE: This study aimed to evaluate the expression level of anti-apoptotic Bcl-2 family proteins in B and T cells in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in relation to disease activity and the effect of various Bcl-2 family inhibitors (BH3 mimetics) as potential treatment. METHODS: We included 14 SLE patients, 12 RA patients, and 13 healthy controls to study anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 expression and cell survival in different B and T cell subsets using stimulation assays and intracellular flow cytometry. Effect of various BH3 mimetics was assessed by cell viability analyses. RESULTS: In SLE, significant differences in Bcl-2 family members were confined to the B cell compartment with decreased induction of Bcl-XL (p ≤ 0.05) and Mcl-1 (p ≤ 0.001) upon CpG stimulation. In RA, we did not observe any differences in expression levels of Bcl-2 family proteins. Expression patterns did not correlate with disease activity apart from decreased induction of Mcl-1 in B cells in active SLE. After in vitro stimulation with CpG, plasmablasts were more viable after treatment with three different BH3 mimetics compared to naïve or memory B cells in control and patient cells. After activation, Mcl-1 inhibition was most effective in reducing plasmablast and T cell viability, however, less in patients than controls. CONCLUSION: Our study provides evidence for the increased differential expression pattern of Bcl-2 family members in B and T cell subsets of patients with SLE compared to controls. Tested BH3 mimetics showed higher efficacy in controls compared to both autoimmune diseases, though nonsignificant due to low patient numbers.

Dysregulated endothelial cell markers in systemic lupus erythematosus: a systematic review and meta-analysis.

OBJECTIVES: To perform a systematic literature review and meta-analysis on endothelial cell (EC) markers that are involved and dysregulated in systemic lupus erythematosus (SLE) in relation to disease activity, as EC dysregulation plays a major role in the development of premature atherosclerosis in SLE. METHODS: Search terms were entered into Embase, MEDLINE, Web of Science, Google Scholar and Cochrane. Inclusion criteria were 1) studies published after 2000 reporting measurements of EC markers in serum and/or plasma of SLE patients (diagnosed according to ACR/SLICC criteria), 2) English language peer reviewed articles, and 3) disease activity measurement. For meta-analysis calculations, the Meta-Essentials tool by Erasmus Research Institute and of Management (ERIM) was used. Only those EC markers, which were 1) reported in at least two articles and 2) reported a correlation coefficient (i.e. Spearman's rank or Pearson's) between the measured levels of the EC marker and disease activity were included. For meta-analyses, a fixed effect model was used. RESULTS: From 2133 hits, 123 eligible articles were selected. The identified SLE-related endothelial markers were involved in EC activation, EC apoptosis, disturbed angiogenesis, defective vascular tone control, immune dysregulation and coagulopathy. Meta-analyses of primarily cross-sectional studies showed significant associations between marker levels and disease activity for the following endothelial markers: Pentraxin-3, Thrombomodulin, VEGF, VCAM-1, ICAM-1, IP-10 and MCP-1. Dysregulated EC markers without associations with disease activity were: Angiopoeitin-2, vWF, P-Selectin, TWEAK and E-Selectin. CONCLUSIONS: We provide a complete literature overview for dysregulated EC markers in SLE comprising a wide range of different EC functions. SLE-induced EC marker dysregulation was seen with, but also without, association with disease activity. This study provides some clarity in the eminent complex field of EC markers as biomarkers for SLE. Longitudinal data on EC markers in SLE are now needed to guide us more in unravelling the pathophysiology of premature atherosclerosis and cardiovascular events in SLE patients.