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1.  Type I Interferon-Mediated Skewing of the Serotonin Synthesis Is Associated with Severe Disease in Systemic Lupus Erythematosus 
PLoS ONE  2015;10(4):e0125109.
Serotonin, a highly pro-inflammatory molecule released by activated platelets, is formed by tryptophan. Tryptophan is also needed in the production of kynurenine, a process mediated by the type I interferon (IFN)-regulated rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO). The aim of this study was to investigate levels of serotonin in patients with the autoimmune disease systemic lupus erythematosus (SLE), association to clinical phenotype and possible involvement of IDO in regulation of serotonin synthesis. Serotonin levels were measured in serum and plasma from patients with SLE (n=148) and healthy volunteers (n=79) by liquid chromatography and ELISA, as well as intracellularly in platelets by flow cytometry. We found that SLE patients had decreased serotonin levels in serum (p=0.01) and platelets (p<0.0001) as compared to healthy individuals. SLE patients with ongoing type I IFN activity, as determined by an in-house reporter assay, had decreased serum levels of serotonin (p=0.0008) as well as increased IDO activity (p<0.0001), as determined by the kynurenine/tryptophan ratio measured by liquid chromatography. Furthermore, SLE sera induced IDO expression in WISH cells in a type I IFN-dependent manner (p=0.008). Also platelet activation contributed to reduce overall availability of serotonin levels in platelets and serum (p<0.05). Decreased serum serotonin levels were associated with severe SLE with presence of anti-dsDNA antibodies and nephritis. In all, reduced serum serotonin levels in SLE patients were related to severe disease phenotype, including nephritis, suggesting involvement of important immunopathological processes. Further, our data suggest that type I IFNs, present in SLE sera, are able to up-regulate IDO expression, which may lead to decreased serum serotonin levels.
PMCID: PMC4405357  PMID: 25897671
2.  Plasma levels of galectin-3-binding protein reflect type I interferon activity and are increased in patients with systemic lupus erythematosus 
Lupus Science & Medicine  2014;1(1):e000026.
Simple measures of type I interferon (IFN) activity constitute highly attractive biomarkers in systemic lupus erythematosus (SLE). We explore galectin-3-binding protein (G3BP) as a novel measure of type I IFN activity and serum/plasma biomarker in large independent cohorts of patients with SLE and controls.
Serum and plasma G3BP concentrations were quantified using ELISA. Type I IFN activity was assessed by Mx1 reporter gene expression assays and correlated to serum G3BP concentrations (SLE-IFN-α, n=26 and healthy controls (HCs), n=10). Plasma G3BP concentrations in the SLE-Denmark (DK) (n=70) and SLE-Sweden (SE) (n=68) cohorts were compared with the HC-DK (n=47) and HC-SE (n=50) cohorts and patients with systemic sclerosis (n=111). In 15 patients with SLE, serum G3BP in consecutive samples was correlated to disease activity. Correlation analysis between G3BP, clinical parameters including disease activity in the four SLE cohorts was performed.
G3BP concentrations correlated significantly with the IFN-α reporter gene assay (r=0.56, p=0.0005) and with IFN-α gene expression scores (r=0.54, p=0.0002). Plasma concentrations were significantly increased in the SLE-DK and SLE-SE cohorts compared with HCs and patients with systemic sclerosis (p<0.0001 and p=0.0009). G3BP concentrations correlated with disease activity measures in the SLE-DK- and SLE-IFN-α cohorts (p=0.0004 and p=0.05) but not in the SLE-SE cohort (p=0.98). Markedly temporal variation was observed in G3BP levels in the consecutive SLE-samples and was significantly associated with changes in disease activity (r=0.44, p=0.014).
G3BP plasma levels reflect type I IFN activity and are increased in SLE. Associations with disease activity or clinical manifestations are uncertain. This study highlights G3BP as a convenient measure of type I IFN-dependent gene activation.
PMCID: PMC4246916  PMID: 25452879
Systemic Lupus Erythematosus; Systemic Sclerosis; Interferon; Lupus Nephritis; Autoantibodies
3.  Platelet Activation and Anti-Phospholipid Antibodies Collaborate in the Activation of the Complement System on Platelets in Systemic Lupus Erythematosus 
PLoS ONE  2014;9(6):e99386.
Anti-phospholipid (aPL) antibodies are important contributors to development of thrombosis in patients with the autoimmune rheumatic disease systemic lupus erythematosus (SLE). The underlying mechanism of aPL antibody-mediated thrombosis is not fully understood but existing data suggest that platelets and the complement system are key components. Complement activation on platelets is seen in SLE patients, especially in patients with aPL antibodies, and has been related to venous thrombosis and stroke. The aim of this study was to investigate if aPL antibodies could support classical pathway activation on platelets in vitro as well as in SLE patients. Furthermore, we investigated if complement deposition on platelets was associated with vascular events, either arterial or venous, when the data had been adjusted for traditional cardiovascular risk factors. Finally, we analyzed if platelet complement deposition, both C1q and C4d, was specific for SLE. We found that aPL antibodies supported C4d deposition on platelets in vitro as well as in SLE patients (p = 0.001 and p<0.05, respectively). Complement deposition on platelets was increased in SLE patients when compared with healthy individuals (p<0.0001). However, high levels of C4d deposition and a pronounced C1q deposition were also seen in patients with rheumatoid arthritis and systemic sclerosis. In SLE, C4d deposition on platelets was associated with platelet activation, complement consumption, disease activity and venous (OR = 5.3, p = 0.02), but not arterial, thrombosis, observations which were independent of traditional cardiovascular risk factors. In conclusion, several mechanisms operate in SLE to amplify platelet complement deposition, of which aPL antibodies and platelet activation were identified as important contributors in this investigation. Complement deposition on platelets was identified as a marker of venous, but not arterial thrombosis, in SLE patients independently of traditional risk factors and aPL antibodies. Further studies are needed to elucidate the role of complement deposition on platelets in development of venous thrombosis.
PMCID: PMC4055750  PMID: 24922069
4.  Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus 
Arthritis Research & Therapy  2014;16(3):R120.
Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE.
The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10−D16low in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR.
SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10−CD16low phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation.
Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.
PMCID: PMC4075132  PMID: 24902963
5.  Clinical phenotype associations with various types of anti-dsDNA antibodies in patients with recent onset of rheumatic symptoms. Results from a multicentre observational study 
Lupus Science & Medicine  2014;1(1):e000007.
Despite anti-dsDNA antibodies constitute a wide range of specificities, they are considered as the hallmark for systemic lupus erythematosus (SLE).
To identify clinical phenotypes associated with anti-dsDNA antibodies, independently of any clinical diagnoses.
Patients with recent onset of any rheumatic symptoms were screened for antinuclear antibodies (ANA). All ANA-positive and matching ANA-negative patients were examined, and their clinical phenotypes were registered, using a systematic chart formulated after consensus between the participating centres. All patients were tested for different anti-dsDNA antibody specificities with assays habitually used in each participating laboratory. Crithidia Luciliae Immuno Fluorescence Test (CLIFT) was performed three times (with two different commercial kits); solid and solution phase ELISA were performed four times. Associations between clinical phenotypes and results of anti-dsDNA assays were evaluated by linear regression analysis (LRA) and principal component analysis (PCA).
Totally, 292 ANA-positive and 292 matching ANA-negative patients were included in the study. A full dataset for statistical analysis was obtained in 547 patients. Anti-dsDNA antibodies were most frequently detected by ELISA. LRA showed that overall positivity of anti-dsDNA antibodies was associated with proteinuria and pleuritis. Alopecia was significantly associated only with CLIFT-positivity. Besides confirming the same findings, PCA showed that combined positivity of CLIFT and ELISA was also associated with lymphopenia.
Our results show that different anti-dsDNA antibody specificities are associated with nephropathy, pleuritis, alopecia and lymphopenia, regardless of the diagnosis. It may challenge the importance of anti-dsDNA antibodies as a diagnostic hallmark for SLE.
PMCID: PMC4225731  PMID: 25396058
Autoantibodies; Autoimmunity; Systemic Lupus Erythematosus; Lupus Nephritis; Autoimmune Diseases
6.  Effects of prostacyclin on the early inflammatory response in patients with traumatic brain injury-a randomised clinical study 
SpringerPlus  2014;3:98.
Objective and design
A prospective, randomised, double-blinded, clinical trial was performed at a level 1 trauma centre to determine if a prostacyclin analogue, epoprostenol (Flolan®), could attenuate systemic inflammatory response in patients with severe traumatic brain injury (TBI).
46 patients with severe TBI, randomised to epoprostenol (n = 23) or placebo (n = 23).
Epoprostenol, 0.5 ng · kg-1 · min-1, or placebo (saline) was given intravenously for 72 hours and then tapered off over the next 24 hours.
Interleukin-6 (IL-6), interleukin-8 (IL-8), soluble intracellular adhesion molecule-1 (sICAM-1), C-reactive protein (CRP), and asymmetric dimethylarginine (ADMA) levels were measured over five days. Measurements were made at 24 h intervals ≤24 h after TBI to 97–120 h after TBI.
A significantly lower CRP level was detected in the epoprostenol group compared to the placebo group within 73–96 h (p = 0.04) and within 97–120 h (p = 0.008) after trauma. IL-6 within 73–96 h after TBI was significantly lower in the epoprostenol group compared to the placebo group (p = 0.04). ADMA was significantly increased within 49–72 h and remained elevated, but there was no effect of epoprostenol on ADMA levels. No significant differences between the epoprostenol and placebo groups were detected for IL-8 or sICAM-1.
Administration of the prostacyclin analogue epoprostenol significantly decreased CRP and, to some extent, IL-6 levels in patients with severe TBI compared to placebo. These findings indicate an interesting option for treatment of TBI and warrants future larger studies.
Trial registration Identifier, NCT01363583
PMCID: PMC3942562  PMID: 24600548
Traumatic brain injury; Epoprostenol; Systemic inflammatory response; Cytokines; Asymmetric dimethylarginine
7.  Association of STAT4 Polymorphism with Severe Renal Insufficiency in Lupus Nephritis 
PLoS ONE  2013;8(12):e84450.
Lupus nephritis is a cause of significant morbidity in systemic lupus erythematosus (SLE) and its genetic background has not been completely clarified. The aim of this investigation was to analyze single nucleotide polymorphisms (SNPs) for association with lupus nephritis, its severe form proliferative nephritis and renal outcome, in two Swedish cohorts. Cohort I (n = 567 SLE cases, n =  512 controls) was previously genotyped for 5676 SNPs and cohort II (n = 145 SLE cases, n = 619 controls) was genotyped for SNPs in STAT4, IRF5, TNIP1 and BLK.
Case-control and case-only association analyses for patients with lupus nephritis, proliferative nephritis and severe renal insufficiency were performed. In the case-control analysis of cohort I, four highly linked SNPs in STAT4 were associated with lupus nephritis with genome wide significance with p = 3.7×10−9, OR 2.20 for the best SNP rs11889341. Strong signals of association between IRF5 and an HLA-DR3 SNP marker were also detected in the lupus nephritis case versus healthy control analysis (p <0.0001). An additional six genes showed an association with lupus nephritis with p <0.001 (PMS2, TNIP1, CARD11, ITGAM, BLK and IRAK1). In the case-only meta-analysis of the two cohorts, the STAT4 SNP rs7582694 was associated with severe renal insufficiency with p  = 1.6×10−3 and OR 2.22. We conclude that genetic variations in STAT4 predispose to lupus nephritis and a worse outcome with severe renal insufficiency.
PMCID: PMC3873995  PMID: 24386384
8.  Contact activation products are new potential biomarkers to evaluate the risk of thrombotic events in systemic lupus erythematosus 
Arthritis Research & Therapy  2013;15(6):R206.
Patients with systemic lupus erythematosus (SLE) have persistent platelet activation and an increased risk of thrombotic events, which cannot be accounted for by traditional cardiovascular risk factors. Factor (F)XII has a potentially important role in thrombus formation and is triggered by activated platelets. We therefore asked whether the contact system is involved in inflammation and vascular disease (VD) in SLE.
Fibrin clots were incubated with purified FXII or whole blood, and the activation and regulation of FXII were studied. Plasma from SLE patients with (n = 31) or without (n = 38) previous VD and from matched healthy controls (n = 68) were analyzed for the presence of complexes formed between contact system enzymes and antithrombin (AT) or C1 inhibitor (C1INH) and evaluated with regard to clinical data and laboratory parameters.
Fibrin clots elicited FXII activation and acted as co-factors for AT. In clotting plasma, the levels of FXIIa-AT increased, and FXIIa-C1INH decreased. A similar reciprocal relationship existed in SLE patients. FXIIa-AT was elevated in the SLE patients with a history of VD, while the corresponding levels of factor FXIIa-C1INH were significantly decreased. FXIIa-AT correlated strongly with platelet parameters. The odds ratio for VD among the SLE patients was 8.9 if they had low levels of FXIIa-C1INH, 6.1 for those with high levels of FXIIa-AT, and increased to 23.4 for those with both decreased levels of FXIIa-C1INH and increased levels of FXIIa-AT.
Activation of FXII is elicited by fibrin during thrombotic reactions in vitro and in vivo, and fibrin acts as a heparin-like co-factor and regulates AT. Patients with SLE had altered levels of FXIIa-serpin complexes, supporting that the contact system is involved in this disease. FXIIa-serpin complexes are strongly associated with previous VD in SLE patients, suggesting that these complexes are potential biomarkers for monitoring and assessing the risk of thrombotic events in SLE.
PMCID: PMC3979000  PMID: 24299152
9.  Genes identified in Asian SLE GWASs are also associated with SLE in Caucasian populations 
Recent genome-wide association studies (GWASs) conducted in Asian populations have identified novel risk loci for systemic lupus erythematosus (SLE). Here, we genotyped 10 single-nucleotide polymorphisms (SNPs) in eight such loci and investigated their disease associations in three independent Caucasian SLE case–control cohorts recruited from Sweden, Finland and the United States. The disease associations of the SNPs in ETS1, IKZF1, LRRC18-WDFY4, RASGRP3, SLC15A4, TNIP1 and 16p11.2 were replicated, whereas no solid evidence of association was observed for the 7q11.23 locus in the Caucasian cohorts. SLC15A4 was significantly associated with renal involvement in SLE. The association of TNIP1 was more pronounced in SLE patients with renal and immunological disorder, which is corroborated by two previous studies in Asian cohorts. The effects of all the associated SNPs, either conferring risk for or being protective against SLE, were in the same direction in Caucasians and Asians. The magnitudes of the allelic effects for most of the SNPs were also comparable across different ethnic groups. On the contrary, remarkable differences in allele frequencies between Caucasian and Asian populations were observed for all associated SNPs. In conclusion, most of the novel SLE risk loci identified by GWASs in Asian populations were also associated with SLE in Caucasian populations. We observed both similarities and differences with respect to the effect sizes and risk allele frequencies across ethnicities.
PMCID: PMC3746253  PMID: 23249952
systemic lupus erythematosus; genetic-association study; Asian; Caucasian
10.  Degradation of neutrophil extracellular traps co-varies with disease activity in patients with systemic lupus erythematosus 
The ability to degrade neutrophil extracellular traps (NETs) is reduced in a subset of patients with systemic lupus erythematosus (SLE). NETs consist of chromatin covered with antimicrobial enzymes and are normally degraded by DNase-I, an enzyme which is known to have reduced activity in SLE. Decreased ability to degrade NETs is associated with disease activity. In the current study we investigated how the ability of serum from SLE patients to degrade NETs varies during the course of SLE as well as what impact this may have for the clinical phenotype of SLE.
Serum from 69 patients with SLE, included in a prospective study, was taken every 60 days for a median of 784 days. The ability of serum to degrade NETs was determined and associated with clinical parameters occurring before and at the time of sampling, as well as after sampling by using conditional logistic regression.
As many as 41% of all patients in the study showed decreased ability to degrade NETs at least once, but with a median of 20% of all time points. Decreased degradation was associated with manifestations of glomerulonephritis as well as low complement levels and elevated levels of antibodies directed against histones and DNA. Furthermore, the odds ratio for the patient to develop alopecia and fever after an episode of decreased NETs degradation was increased by four to five times compared to normal.
Decreased degradation of NETs is associated with clinical manifestations in SLE and may contribute to disease pathogenesis. Potential therapeutics restoring the ability to degrade NETs could be beneficial for certain patients with SLE.
PMCID: PMC3978901  PMID: 23945056
Systemic lupus erythematosus; neutrophil extracellular traps; degradation; glomerulonephritis; prospective study
11.  Decreased Neutrophil Apoptosis in Quiescent ANCA-Associated Systemic Vasculitis 
PLoS ONE  2012;7(3):e32439.
ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis, accumulation of unscavenged apoptotic and necrotic neutrophils in perivascular tissues. Dysregulation of neutrophil cell death may contribute directly to the pathogenesis of AASV.
Neutrophils from Healthy Blood Donors (HBD), patients with AASV most in complete remission, Polycythemia Vera (PV), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and renal transplant recipients (TP) were incubated in vitro, and the rate of spontaneous apoptosis was measured by FACS. Plasma levels of cytokines and sFAS were measured with cytometric bead array and ELISA. Expression of pro/anti-apoptotic factors, transcription factors C/EBP-α, C/EBP-β and PU.1 and inhibitors of survival/JAK2-pathway were measured by real-time-PCR.
AASV, PV and RA neutrophils had a significantly lower rate of apoptosis compared to HBD neutrophils (AASV 50±14% vs. HBD 64±11%, p<0.0001). In RA but not in AASV and PV, low apoptosis rate correlated with increased plasma levels of GM-CSF and high mRNA levels of anti-apoptotic factors Bcl-2A1 and Mcl-1. AASV patients had normal levels of G-CSF, GM-CSF and IL-3. Both C/EBP-α, C/EBP-β were significantly higher in neutrophils from AASV patients than HBD. Levels of sFAS were significantly higher in AASV compared to HBD.
Neutrophil apoptosis rates in vitro are decreased in AASV, RA and PV but mechanisms seem to differ. Increased mRNA levels of granulopoiesis-associated transcription factors and increased levels of sFAS in plasma were observed in AASV. Additional studies are required to define the mechanisms behind the decreased apoptosis rates, and possible connections with accumulation of dying neutrophils in regions of vascular lesions in AASV patients.
PMCID: PMC3293802  PMID: 22403660
12.  Serum COMP-C3b complexes in rheumatic diseases and relation to anti-TNF-α treatment 
Cartilage oligomeric matrix protein (COMP) is found at elevated concentrations in sera of patients with joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). We recently showed that COMP activates complement via the alternative pathway and that COMP-C3b complexes are present in sera of RA patients, but not in healthy controls. We now set out to elaborate on the information provided by this marker in a variety of diseases and larger patient cohorts.
COMP-C3b levels in sera were measured by using an enzyme-linked immunosorbent assay (ELISA) capturing COMP and detecting C3b. Serum COMP was measured by using ELISA.
COMP-C3b levels were significantly elevated in patients with RA as well as in systemic lupus erythematosus (SLE), compared with healthy controls. SLE patients with arthritis had significantly higher COMP-C3b levels than did those without. COMP-C3b was furthermore elevated in patients with ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, systemic sclerosis, and OA. COMP-C3b did not correlate with COMP in any of the patient groups. COMP-C3b correlated with disease activity in RA, but not in other diseases. COMP-C3b levels in RA patients decreased on treatment with tumor necrosis factor (TNF)-α inhibitors, whereas the levels increased in patients with AS or PsA. The changes of COMP-C3b did not parallel the changes of C-reactive protein (CRP).
COMP-C3b levels are elevated in several rheumatologic diseases and correlate with inflammatory measures in RA. COMP-C3b levels in RA decrease during TNF-α inhibition differently from those of CRP, suggesting that formation of COMP-C3b relates to disease features not reflected by general inflammation measures.
PMCID: PMC3392805  PMID: 22264230
13.  Mutations in genes encoding complement inhibitors CD46 and CFH affect the age at nephritis onset in patients with systemic lupus erythematosus 
Arthritis Research & Therapy  2011;13(6):R206.
Inherited deficiencies of several complement components strongly predispose to systemic lupus erythematosus (SLE) while deficiencies of complement inhibitors are found in kidney diseases such as atypical hemolytic uremic syndrome (aHUS).
The exons of complement inhibitor genes CD46 and CFH (factor H) were fully sequenced using the Sanger method in SLE patients with nephritis originating from two cohorts from southern and mid Sweden (n = 196). All identified mutations and polymorphisms were then analyzed in SLE patients without nephritis (n = 326) and in healthy controls (n = 523).
We found nonsynonymous, heterozygous mutations in CFH in 6.1% patients with nephritis, in comparison with 4.0% and 5.4% in patients without nephritis and controls, respectively. No associations of SLE or nephritis with common variants in CFH (V62I/Y402H/E936D) were found. Furthermore, we found two nonsynonymous heterozygous mutations in CD46 in SLE patients but not in controls. The A353V polymorphism, known to affect function of CD46, was found in 6.6% of nephritis patients versus 4.9% and 6.1% of the non-nephritis SLE patients and controls. The presence of mutations in CD46 and CFH did not predispose to SLE or nephritis but was associated with earlier onset of nephritis. Furthermore, we found weak indications that there is one protective and one risk haplotype predisposing to nephritis composed of several polymorphisms in noncoding regions of CD46, which were previously implicated in aHUS.
SLE nephritis is not associated with frequent mutations in CFH and CD46 as found in aHUS but these may be modifying factors causing earlier onset of nephritis.
PMCID: PMC3334659  PMID: 22171659
14.  Increased plasma levels of the soluble Mer tyrosine kinase receptor in systemic lupus erythematosus relate to disease activity and nephritis 
Mer and Tyro3 are receptor tyrosine kinases important for the phagocytosis of apoptotic cells. Together with Axl, they constitute the TAM receptor family. These receptors can be shed from the cell membrane and their soluble extracellular regions can be found in plasma. The objective of this study was to elucidate whether the plasma levels of soluble Mer (sMer) and Tyro3 (sTyro3) were increased in systemic lupus erythematosis (SLE), rheumatoid arthritis (RA), or critical limb ischemia (CLI).
ELISA kits were used to test plasma concentrations in controls and in patients with SLE, RA or CLI.
Increased levels of, in particular, sMer and, to some extent, sTyro3, were found in patients with SLE or RA, but not in patients with CLI. Patients with SLE demonstrated the highest sMer levels and there was a strong correlation to higher SLE disease activity score (SLEDAI). In contrast, in patients with RA, the sMer levels did not correlate with the disease activity score (DAS). In SLE, sMer levels were particularly high in those with lupus nephritis, patients who also had decreased C1q levels and increased titers of anti-DNA antibodies. After therapy, the plasma concentrations of sMer decreased in parallel to the decrease in SLEDAI score.
The plasma concentrations of sMer and sTyro3 were significantly increased in patients with active SLE and RA, suggesting the TAM receptor shedding was affected by these autoimmune diseases. In particular, sMer was increased in SLE, the plasma levels of sMer reflecting disease activity.
PMCID: PMC3132057  PMID: 21496228
15.  Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus 
Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9.
Flow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively.
Cell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9.
Patients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE.
PMCID: PMC3132055  PMID: 21492422
16.  A candidate gene study of the type I interferon pathway implicates IKBKE and IL8 as risk loci for SLE 
Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease in which the type I interferon pathway has a crucial role. We have previously shown that three genes in this pathway, IRF5, TYK2 and STAT4, are strongly associated with risk for SLE. Here, we investigated 78 genes involved in the type I interferon pathway to identify additional SLE susceptibility loci. First, we genotyped 896 single-nucleotide polymorphisms in these 78 genes and 14 other candidate genes in 482 Swedish SLE patients and 536 controls. Genes with P<0.01 in the initial screen were then followed up in 344 additional Swedish patients and 1299 controls. SNPs in the IKBKE, TANK, STAT1, IL8 and TRAF6 genes gave nominal signals of association with SLE in this extended Swedish cohort. To replicate these findings we extracted data from a genomewide association study on SLE performed in a US cohort. Combined analysis of the Swedish and US data, comprising a total of 2136 cases and 9694 controls, implicates IKBKE and IL8 as SLE susceptibility loci (Pmeta=0.00010 and Pmeta=0.00040, respectively). STAT1 was also associated with SLE in this cohort (Pmeta=3.3 × 10−5), but this association signal appears to be dependent of that previously reported for the neighbouring STAT4 gene. Our study suggests additional genes from the type I interferon system in SLE, and highlights genes in this pathway for further functional analysis.
PMCID: PMC3060320  PMID: 21179067
systemic lupus erythematosus; type I interferon system; candidate gene study; single nucleotide polymorphism; IKBKE; IL8
17.  Serum Protein Profiling of Systemic Lupus Erythematosus and Systemic Sclerosis Using Recombinant Antibody Microarrays* 
Molecular & Cellular Proteomics : MCP  2011;10(5):M110.005033.
Systemic lupus erythematosus (SLE) and systemic sclerosis (SSc) are two severe autoimmune connective tissue diseases. The fundamental knowledge about their etiology is limited and the conditions display complex pathogenesis, multifaceted presentations, and unpredictable courses. Despite significant efforts, the lack of fully validated biomarkers enabling diagnosis, classification, and monitoring of disease activity represents significant unmet clinical needs. In this discovery study, we have for the first time used recombinant antibody microarrays for miniaturized, multiplexed serum protein profiling of SLE and SSc, targeting mainly immunoregulatory proteins. The data showed that several candidate SLE-associated multiplexed serum biomarker signatures were delineated, reflecting disease (diagnosis), disease severity (phenotypic subsets), and disease activity. Selected differentially expressed markers were validated using orthogonal assays and a second, independent patient cohort. Further, biomarker signatures differentiating SLE versus SSc were demonstrated, and the observed differences increased with severity of SLE. In contrast, the data showed that the serum profiles of SSc versus healthy controls were more similar. Hence, we have shown that affinity proteomics could be used to de-convolute crude, nonfractionated serum proteomes, extracting molecular portraits of SLE and SSc, further enhancing our fundamental understanding of these complex autoimmune conditions.
PMCID: PMC3098590  PMID: 21350050
18.  Plasma concentrations of Gas6 and sAxl correlate with disease activity in systemic lupus erythematosus 
Rheumatology (Oxford, England)  2011;50(6):1064-1069.
Objectives. SLE is a systemic autoimmune disease with an annual incidence of 3.8 per 100 000. Several pathogenic mechanisms are believed to be operating in SLE, including an impaired clearance of apoptotic cells, activation of the type I IFN pathway and generation of autoimmune leucocytes. Growth arrest-specific protein 6 (Gas6) and its receptor Axl are known to regulate inflammation and may be implicated in lupus pathogenesis. We have recently developed immunological methods to quantify the vitamin-K-dependent protein Gas6 and its soluble receptor sAxl in human plasma, which we have used to investigate the role of Gas6 and soluble Axl in SLE.
Methods. We have investigated the relation between the plasma concentrations of Gas6 and sAxl and disease activity and specific symptoms in 96 SLE patients.
Results. Gas6 and sAxl concentrations correlated with SLEDAI (r = 0.48, P < 0.001 and r = 0.39, P < 0.001, respectively). Furthermore, concentrations of Gas6 and sAxl correlated with ESR and CRP and inversely with haemoglobin levels. Gas6 and sAxl concentrations were significantly higher in patients with anti-DNA antibodies, leucopenia and GN.
Conclusion. The plasma concentrations of Gas6 and sAxl vary with disease activity in SLE, in particular GN, and may have a role in lupus pathogenesis. Furthermore, Gas6 and sAxl may be of use as biomarkers of disease activity.
PMCID: PMC3093930  PMID: 21278074
Systemic lupus erythematosus; Growth arrest-specific protein 6; Axl; Receptor tyrosine kinase; Vitamin K; Inflammation; Autoimmunity
19.  A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus 
Nature genetics  2009;41(11):1228-1233.
Genome-wide association studies have recently identified at least 15 susceptibility loci for systemic lupus erythematosus (SLE). To confirm additional risk loci, we selected SNPs from 2,466 regions that showed nominal evidence of association to SLE (P < 0.05) in a genome-wide study and genotyped them in an independent sample of 1,963 cases and 4,329 controls. This replication effort identified five new SLE susceptibility loci (P < 5 × 10−8): TNIP1 (odds ratio (OR) = 1.27), PRDM1 (OR = 1.20), JAZF1 (OR = 1.20), UHRF1BP1 (OR = 1.17) and IL10 (OR = 1.19). We identified 21 additional candidate loci with P ≤ 1 × 10−5. A candidate screen of alleles previously associated with other autoimmune diseases suggested five loci (P < 1 × 10−3) that may contribute to SLE: IFIH1, CFB, CLEC16A, IL12B and SH2B3. These results expand the number of confirmed and candidate SLE susceptibility loci and implicate several key immunologic pathways in SLE pathogenesis.
PMCID: PMC2925843  PMID: 19838195
20.  A risk haplotype of STAT4 for systemic lupus erythematosus is over-expressed, correlates with anti-dsDNA and shows additive effects with two risk alleles of IRF5 
Human Molecular Genetics  2008;17(18):2868-2876.
Systemic lupus erythematosus (SLE) is the prototype autoimmune disease where genes regulated by type I interferon (IFN) are over-expressed and contribute to the disease pathogenesis. Because signal transducer and activator of transcription 4 (STAT4) plays a key role in the type I IFN receptor signaling, we performed a candidate gene study of a comprehensive set of single nucleotide polymorphism (SNPs) in STAT4 in Swedish patients with SLE. We found that 10 out of 53 analyzed SNPs in STAT4 were associated with SLE, with the strongest signal of association (P = 7.1 × 10−8) for two perfectly linked SNPs rs10181656 and rs7582694. The risk alleles of these 10 SNPs form a common risk haplotype for SLE (P = 1.7 × 10−5). According to conditional logistic regression analysis the SNP rs10181656 or rs7582694 accounts for all of the observed association signal. By quantitative analysis of the allelic expression of STAT4 we found that the risk allele of STAT4 was over-expressed in primary human cells of mesenchymal origin, but not in B-cells, and that the risk allele of STAT4 was over-expressed (P = 8.4 × 10−5) in cells carrying the risk haplotype for SLE compared with cells with a non-risk haplotype. The risk allele of the SNP rs7582694 in STAT4 correlated to production of anti-dsDNA (double-stranded DNA) antibodies and displayed a multiplicatively increased, 1.82-fold risk of SLE with two independent risk alleles of the IRF5 (interferon regulatory factor 5) gene.
PMCID: PMC2525501  PMID: 18579578
21.  Use of Serum or Buffer-Changed EDTA-Plasma in a Rapid, Inexpensive, and Easy-To-Perform Hemolytic Complement Assay for Differential Diagnosis of Systemic Lupus Erythematosus and Monitoring of Patients with the Disease▿  
Clinical and Vaccine Immunology  2007;14(5):549-555.
We previously described a simplified quantitative hemolytic assay for classical pathway (CP) hemolytic function in serum that has been shown to correlate with the 50% hemolytic complement (CH50) assay. In the present study, we used this assay to compare CP functions; plasma levels of C3, C4, and C3dg; and ratios of C3dg to C3 in healthy individuals and patients with systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA) with different degrees of complement activation. A significant depression in CP function and levels of C4 and C3 and increased C3dg levels and C3dg/C3 ratios were observed in the SLE patients. In patients with RA, CP function was normal, whereas C3, C4, and C3dg levels and the C3dg/C3 ratio were elevated. The SLE results are compatible with systemic complement consumption, whereas the RA data suggest an acute-phase reaction with a normal C3 catabolic rate. To facilitate the handling of patient samples, we also developed a method to restore the hemolytic function of EDTA-plasma by transferring it to Veronal-buffered saline containing the thrombin inhibitor lepirudin. This process inhibits coagulation and enables complement activation, allowing a longer time lag between sample harvesting and testing. These results, combined with previous correlation studies, suggest that the CP hemolytic assay can effectively replace the CH50 assay for routine SLE differential diagnosis and monitoring of disease activity.
PMCID: PMC1865633  PMID: 17344343
22.  Microarray image analysis: background estimation using quantile and morphological filters 
BMC Bioinformatics  2006;7:96.
In a microarray experiment the difference in expression between genes on the same slide is up to 103 fold or more. At low expression, even a small error in the estimate will have great influence on the final test and reference ratios. In addition to the true spot intensity the scanned signal consists of different kinds of noise referred to as background. In order to assess the true spot intensity background must be subtracted. The standard approach to estimate background intensities is to assume they are equal to the intensity levels between spots. In the literature, morphological opening is suggested to be one of the best methods for estimating background this way.
This paper examines fundamental properties of rank and quantile filters, which include morphological filters at the extremes, with focus on their ability to estimate between-spot intensity levels. The bias and variance of these filter estimates are driven by the number of background pixels used and their distributions. A new rank-filter algorithm is implemented and compared to methods available in Spot by CSIRO and GenePix Pro by Axon Instruments. Spot's morphological opening has a mean bias between -47 and -248 compared to a bias between 2 and -2 for the rank filter and the variability of the morphological opening estimate is 3 times higher than for the rank filter. The mean bias of Spot's second method,, is between -5 and -16 and the variability is approximately the same as for morphological opening. The variability of GenePix Pro's region-based estimate is more than ten times higher than the variability of the rank-filter estimate and with slightly more bias. The large variability is because the size of the background window changes with spot size. To overcome this, a non-adaptive region-based method is implemented. Its bias and variability are comparable to that of the rank filter.
The performance of more advanced rank filters is equal to the best region-based methods. However, in order to get unbiased estimates these filters have to be implemented with great care. The performance of morphological opening is in general poor with a substantial spatial-dependent bias.
PMCID: PMC1525206  PMID: 16504173
23.  Analysis of HLA DR, HLA DQ, C4A, FcγRIIa, FcγRIIIa, MBL, and IL-1Ra allelic variants in Caucasian systemic lupus erythematosus patients suggests an effect of the combined FcγRIIa R/R and IL-1Ra 2/2 genotypes on disease susceptibility 
Arthritis Research & Therapy  2004;6(6):R557-R562.
Dysfunction in various parts of immune defence, such as immune response, immune complex clearance, and inflammation, has an impact on pathogenesis in systemic lupus erythematosus (SLE). We hypothesised that combinations of common variants of genes involved in these immune functions are associated with susceptibility to SLE. The following variants were analysed: HLA DR3, HLA DQ2, C4AQ0, Fcγ receptor IIa (FcγRIIa) genotype R/R, Fcγ receptor IIIa (FcRγIIIa) genotype F/F, mannan-binding lectin (MBL) genotype conferring a low serum concentration of MBL (MBL-low), and interleukin-1 receptor antagonist (IL-1Ra) genotype 2/2. Polymorphisms were analysed in 143 Caucasian patients with SLE and 200 healthy controls. HLA DR3 in SLE patients was in 90% part of the haplotype HLA DR3-DQ2-C4AQ0, which was strongly associated with SLE (odds ratio [OR] 2.8, 95% CI 1.7–4.5). Analysis of combinations of gene variants revealed that the strong association with SLE for HLA DR3-DQ2-C4AQ0 remained after combination with FcγRIIa R/R, FcγRIIIa F/F, and MBL-low (OR>2). Furthermore, the combination of the FcγRIIa R/R and IL-1Ra 2/2 genotypes yielded a strong correlation with SLE (OR 11.8, 95% CI 1.5–95.4). This study demonstrates that certain combinations of gene variants may increase susceptibility to SLE, suggesting this approach for future studies. It also confirms earlier findings regarding the HLA DR3-DQ2-C4AQ0 haplotype.
PMCID: PMC1064866  PMID: 15535834
Fcγ receptor; HLA; interleukin-1 receptor antagonist; mannan-binding lectin; systemic lupus erythematosus
24.  P2 receptor mRNA expression profiles in human lymphocytes, monocytes and CD34+ stem and progenitor cells 
BMC Immunology  2004;5:16.
Extracellular nucleotides (ATP, ADP, UTP and UDP) exert a wide range of biological effects in blood cells mediated by multiple ionotropic P2X receptors and G protein-coupled P2Y receptors. Although pharmacological experiments have suggested the presence of several P2 receptor subtypes on monocytes and lymphocytes, some results are contradictory. Few physiological functions have been firmly established to a specific receptor subtype, partly because of a lack of truly selective agonists and antagonists. This stimulated us to investigate the expression of P2X and P2Y receptors in human lymphocytes and monocytes with a newly established quantitative mRNA assay for P2 receptors. In addition, we describe for the first time the expression of P2 receptors in CD34+ stem and progenitor cells implicating a potential role of P2 receptors in hematopoietic lineage and progenitor/stem cell function.
Using a quantitative mRNA assay, we assessed the hypothesis that there are specific P2 receptor profiles in inflammatory cells. The P2X4 receptor had the highest expression in lymphocytes and monocytes. Among the P2Y receptors, P2Y12 and P2Y2 had highest expression in lymphocytes, while the P2Y2 and P2Y13 had highest expression in monocytes. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4) in CD34+ stem and progenitor cells.
The most interesting findings were the high mRNA expression of P2Y12 receptors in lymphocytes potentially explaining the anti-inflammatory effects of clopidogrel, P2Y13 receptors in monocytes and a previously unrecognised expression of P2X4 in lymphocytes and monocytes. In addition, for the first time P2 receptor mRNA expression patterns was studied in CD34+ stem and progenitor cells. Several P2 receptors were expressed (P2Y2, P2Y1, P2Y12, P2Y13, P2Y11, P2X1, P2X4), indicating a role in differentiation and proliferation. Thus, it is possible that specific antibodies to P2 receptors could be used to identify progenitors for monocytes, lymphocytes and megakaryocytes.
PMCID: PMC509419  PMID: 15291969
P2 receptor; real-time PCR; lymphocytes; monocytes; CD34+ cells; hematopoietic stem cells
25.  Serum levels of autoantibodies against monomeric C-reactive protein are correlated with disease activity in systemic lupus erythematosus 
Arthritis Research & Therapy  2003;6(2):R87-R94.
This study was performed to investigate the relation between IgG autoantibodies against human C-reactive protein (anti-CRP) and disease activity measures in serial serum samples from 10 patients with systemic lupus erythematosus (SLE), of whom four had active kidney involvement during the study period. The presence of anti-CRP was analysed by enzyme-linked immunosorbent assay. The cut-off for positive anti-CRP test was set at the 95th centile of 100 healthy blood donor sera. Specificity of the anti-CRP antibody binding was evaluated by preincubating patient sera with either native or monomeric CRP. Disease activity was determined by the SLE disease activity index (SLEDAI), serum levels of CRP, anti-DNA antibodies, complement components and blood cell counts. Of 50 serum samples, 20 (40%) contained antibodies reactive with monomeric CRP, and 7 of 10 patients were positive on at least one occasion during the study. All patients with active lupus nephritis were positive for anti-CRP at flare. Frequent correlations between anti-CRP levels and disease activity measures were observed in anti-CRP-positive individuals. Accumulated anti-CRP data from all patients were positively correlated with SLEDAI scores and anti-DNA antibody levels, whereas significant inverse relationships were noted for complement factors C1q, C3 and C4, and for lymphocyte counts. This study confirms the high prevalence of anti-CRP autoantibodies in SLE and that the antibody levels are correlated with clinical and laboratory disease activity measures. This indicates that anti-CRP antibodies might have biological functions of pathogenetic interest in SLE. Further prospective clinical studies and experimental studies on effects mediated by anti-CRP antibodies are warranted.
PMCID: PMC400426  PMID: 15059271
autoantibodies; C-reactive protein; disease activity; SLEDAI; systemic lupus erythematosus

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