Sera Of Systemic Lupus Erythematosus Patients Research Articles

MO430MASS-SPECTROMETRIC IDENTIFICATION OF POST-TRANSLATIONAL GUANIDINYLATED PROTEINS IN THE CONTEXT OF SYSTEMIC LUPUS ERYTHEMATOSUS

Abstract Background and Aims With continuous identification of post-translational modified isoforms of proteins, it is becoming increasingly clear that post-translational modifications limit or modify the biological functions of native proteins are majorly involved in development of various chronic disease. This is mostly due to technically advanced molecular identification and quantification methods, mainly based on mass spectrometry. Mass spectrometry has become one of the most powerful tools for the identification of lipids. Method In this study, we used sophisticated high-resolution mass-spectrometric methods to analyze the soluble ligand of receptor Notch-3, namely the Y-box protein (YB)-1, in serum from systemic lupus erythematosus (SLE) patients. In addition, kidneys of lupus-prone (MRL.lpr) mice were analyzed by mass-spectrometric imaging techniques to identify the underlying pathomechanisms. Serum YB-1 was isolated by chromatographic methods, afterwards digested by trypsin and analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The kidneys were fixed in paraffin, then kidney sections were deparaffinized, tryptic digested and analyzed by mass-spectrometric imaging techniques. Mass-spectrometry of extracellular YB-1 in SLE patient serum revealed post-translational guanidinylation of two lysine’s within the highly conserved cold shock domain (CSD) of the YB-1 protein (YB-1-2G). Patients with increased disease activity and those with active renal involvement (lupus nephritis, LN) had a higher degree of dual-guanidinylation within the CSD. Of note, at least one of these modifications was present in all analyzed LN patients, whereas single-guanidinylated YB-1 was present in only one and double modification in none of the control individuals. Mass-spectrometric imaging analyses specifically localized YB-1-2G and increases Notch-3 expression in kidney sections from MRL.lpr mice. Results The data from this study clearly demonstrate the high potential of high-resolution mass spectrometric methods as well as mass spectrometric imaging techniques to identify pathomechanisms of diseases like SLE/LN.

Untargeted lipidomics reveals specific lipid abnormalities in systemic lupus erythematosus

To identify potential lipid biomarkers by studying changes in the blood lipid profile of patients with systemic lupus erythematosus (SLE) using lipidomics. Serum samples were collected from 115 SLE patients and 115 age- and sex-matched healthy controls (HCs). Lipid profiles were assessed using ultrahigh-performance liquid chromatography coupled with Q Exactive spectrometry, and possible lipid biomarkers were screened and evaluated by univariate and multivariate analyses. Metabolic phenotypes related to SLE disease activity index (SLEDAI) scores were detected in the serum of SLE patients, and these phenotypes indicated the activity of the disease. Alterations in energy metabolism, fatty acid metabolism and other pathways were observed in patients with SLE. Phosphatidylethanolamine (16:0/18:2), lysophosphatidylethanolamine (18:0), and acylcarnitine (11:0) can be used as biomarkers for the clinical diagnosis of SLE, and receiver operating characteristic (ROC) analysis indicated their effectiveness in diagnosing this disease. Our study identified serum biomarkers related to disease activity in patients with SLE, providing a basis for its clinical diagnosis.

Downregulated Serum Exosomal miR-451a Expression Correlates With Renal Damage and Its Intercellular Communication Role in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease characterized by continuous inflammation and the production of autoantibodies. Exosomes, acting as a critical tool for communication between cells, are involved in the pathogenesis of SLE, particularly in inflammation and immune imbalance. In this study, we aimed to extract and confirm the pro-inflammatory effect of serum exosomes in SLE. Then, we attempted to find differentially expressed exosomal microRNAs in the serum of healthy subjects and SLE patients via miRNA microarray analysis and validated the target exosomal microRNA, exosomal miR-451a, which expression level decreased in serum of SLE patients by RT-qPCR. Furtherly, we analyzed the correlation between exosomal miR-451a and disease activity, kidney damage and typing, and traditional medicine therapy. Finally, we investigated the intercellular communication role of exosomal miR-451a in SLE by co-culture assay in vitro. Taken together, our study demonstrated that downregulated serum exosomal miR-451a expression correlated with SLE disease activity and renal damage as well as its intercellular communication role in SLE which provided potential therapeutic strategies.

Anti-Ficolin-2 and Anti-Ficolin-3 Autoantibody Detection by ELISA.

Ficolins are recognition proteins of the lectin pathway of the complement system and also play an important role in innate immunity and in the maintenance of tissue homeostasis. They deserve special attention in the context of autoimmunity since they are involved in the uptake of dying cells. Because the monitoring of systemic lupus erythematosus (SLE) patients is particularly difficult, it is crucial to find new relevant serum biomarkers. The ability to detect autoantibodies in the patients' sera provides a diagnostic and prognostic advantage. We describe in this chapter quantitative enzyme linked immunosorbent assays (ELISA) to detect the presence of autoantibodies targeting ficolin-2 and ficolin-3 in human sera. Recombinant ficolins produced in a mammalian expression system are used as coating antigens. The described in-house ELISAs provide a valuable tool to efficiently quantify anti-ficolin autoantibodies in the sera of SLE patients.

Absence of Anti-Glomerular Basement Membrane Antibodies in 200 Patients With Systemic Lupus Erythematosus With or Without Lupus Nephritis: Results of the GOODLUPUS Study.

IntroductionAnti-glomerular basement membrane (GBM) antibodies are pathogenic antibodies first detected in renal-limited anti-GBM disease and in Goodpasture disease, the latter characterized by rapidly progressive crescentic glomerulonephritis combined with intra-alveolar hemorrhage. Studies have suggested that anti-GBM antibody positivity may be of interest in lupus nephritis (LN). Moreover, severe anti-GBM vasculitis cases in patients with systemic lupus erythematosus (SLE) have been described in the literature, but few studies have assessed the incidence of anti-GBM antibodies in SLE patients.ObjectiveThe main study objective was to determine if positive anti-GBM antibodies were present in the serum of SLE patients with or without proliferative renal damage and compared to a healthy control group.MethodologyThis retrospective study was performed on SLE patients’ sera from a Franco-German European biobank, developed between 2011 and 2014, from 17 hospital centers in the Haut-Rhin region. Patients were selected according to their renal involvement, and matched by age and gender. The serum from healthy voluntary blood donors was also tested. Anti-GBM were screened by fluorescence enzyme immunoassay (FEIA), and then by indirect immunofluorescence (IIF) in case of low reactivity detection (titer >6 U/ml).ResultsThe cohort was composed of 100 SLE patients with proliferative LN (27% with class III, 67% with class IV, and 6% with class V), compared to 100 SLE patients without LN and 100 controls. Patients were mostly Caucasian and met the ACR 1997 criteria and/or the SLICC 2012 criteria. Among the 300 tested sera, no significant levels of anti-GBM antibodies were detected (>10 U/ml) by the automated technique, three sera were found “ambivalent” (>7 U/ml): one in the SLE with LN group and two in the SLE without LN group. Subsequent IIF assays did not detect anti-GBM antibodies.ConclusionAnti-GBM antibodies were not detected in the serum of Caucasian patients with SLE, even in case of renal involvement, a situation favoring the antigenic exposure of glomerular basement membranes. Our results reaffirm the central role of anti-GBM antibodies as a specific diagnostic biomarker for Goodpasture vasculitis and therefore confirm that anti-GBM antibody must not be carried out in patients with SLE (with or without LN) in the absence of disease-suggestive symptoms.

Serum Microrna155 Expression Level in Systemic Lupus Erythematosus Related Peripheral Neuropathy

Background: miRNA-155 (miR-155) became a focus in several studies because of its essential functions in autoimmune diseases and inflammatory responses such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Objective: We aimed in the current study to explore the expression profile of micro RNA-155 in SLE and evaluate its association with the clinical, immunological, and electrophysiological tests of patients with peripheral neuropathy (PN). Patients and methods: Ninety-five recently diagnosed systemic lupus erythematosus (SLE) patients according to 2012 Systemic Lupus International Collaborating Clinics classification criteria were enrolled in addition to 100 controls. Peripheral nerve conduction was evaluated by performing nerve conduction studies (NCSs). The serum miRNA-155 expression profiles were measured using a quantitative real time-polymerase chain reaction (qRT-PCR). Results: Of the 95 SLE patients, PN was present in 35 patients (36.8%). The serum miR-155 expression levels were upregulated the in serum of SLE patients (1.17±0.21) compared to controls (0.826±0.169), P < 0.001. Among SLE groups, patients with PN had a higher level of miR-155 expression (1.24±0.1781) than patients without PN (0.913±0.046), P < 0.001. There was a significantly positive correlation of miR-155 expression levels with The Toronto Clinical Scoring System (TCSS), immunological markers, and electrophysiological tests of median and ulnar nerve. Conclusion: This is the first Egyptian study report that miR-155 expression profile is upregulated in SLE patients especially patients with PN indicating miRNA-155 might be a potential biomarker for the diagnosis and treatment of SLE related PN.

IgGs-Abzymes from the Sera of Patients with Systemic Lupus Erythematosus Hydrolyzed miRNAs.

Background and ObjectivesSystemic lupus erythematosus (SLE) is an inflammatory disease. The sera of SLE patients contain antibodies-abzymes hydrolyzing myelin basic protein (MBP), DNA, nucleotides, and oligosaccharides. The blood of SLE patients contains an increased amount of some specific miRNAs. This study aimed to analyze a possible hydrolysis of eight microRNAs found in the blood of SLE patients with high frequency by blood antibodies-abzymes.Patients and MethodsUsing affinity chromatography of the serum proteins of SLE patients and healthy donors on protein G-Sepharose and following FPLC gel filtration, electrophoretically homogeneous IgG preparations containing no impurities of canonical RNases were obtained. These preparations were used to analyze their activity in the hydrolysis of eight miRNAs.ResultsIt was shown that SLE IgGs hydrolyze very efficiently four neuroregulatory miRNAs (miR-219-2-3p, miR-137, miR-219a-5p, and miR-9-5p) and four immunoregulatory miRNAs (miR-326, miR-21-3p, miR-155-5p, and miR-146a-3p). To demonstrate that the miRNAs hydrolysis is an intrinsic property of SLE IgGs, several rigid criteria were checked. Only some IgGs of healthy donors showed very weak, but reliably detectable activity in the hydrolysis miRNAs. The average activity of SLE patients IgGs according to median values is statistically significant 84.8-fold higher than that of healthy donors. The maximum and comparable average activity (RA) was observed in the hydrolysis of three miRAs: miR-9-5p, miR-155-5p, and miR-326. MiR-9-5p plays an important role in the development of lupus nephritis, while miR-326 activates the production of antibodies by B cells. The major and moderate specific sites of the hydrolysis of each miRNA were revealed. The hydrolysis of eight microRNAs was mostly site specific. Several SLE IgGs hydrolyzed some miRNAs demonstrating a combination of site-specific and non-specific splitting.ConclusionSince inflammatory processes in SLE are associated with the change in miRNAs expression, the decrease in their concentration due to hydrolysis by autoantibodies-abzymes may be important for SLE pathogenesis.

A novel immunochromatographic strips assay for rapid and simple detection of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a complex multi-system autoimmune disease. Detection of anti-nuclear antibodies (ANA) is fundamental for the diagnosis of SLE. In the present study, we found that the level of core fucosylation catalyzed by α1,6-fucosyltransferase (Fut8) is markedly up-regulated on immunoglobulin G (IgG) in the sera of SLE patients detected by Aspergillus oryzae lectin (AOL) blot. In sandwich Dot enzyme-linked immunosorbent assay (Dot-ELISA), the core fucosylation level was also found significantly increased in the sera from SLE patients with a higher ANA titer. To establish a rapid and sensitive laboratory test for the diagnosis of SLE, we prokaryotically expressed AOL and C3-D1-C3-D2-C3 of protein G (SpG3), and generate AOL-conjugated colloid gold immunochromatographic strips (ICS). The detection limit of core fucosylated IgG was 10 μg/mL for AOL-conjugated colloid gold ICS. As well as indirect immunofluorescence, the AOL-conjugated colloid gold ICS showed reliable results in the serum of 39 SLE patients. Our results indicated that the AOL-conjugated colloid gold ICS could serve as a rapid test for the detection of ANA and suspected cases of SLE.

Anti-C1q antibodies in lupus nephritis and their correlation with the disease activity.

Systemic lupus erythematosus (SLE) is an autoimmune connective tissue disorder. Renal involvement usually develops in the first few years of illness and should be detected early by periodic urine analysis and quantitation of proteinuria. The aim of our work was to evaluate the biological marker [anti-complement 1q antibodies (anti-C1q Ab)] in lupus nephritis (LN) patients and its correlation to SLE disease activity. Sixty-five subjects were divided into four groups; Group I: SLE patients with LN (proteinuria >0.5 g/day), Group II: SLE patients without LN (all of them had a lupus flares rather than nephritis; active nonrenal), Group III: SLE patients without any activity (inactive disease), and Group IV: Control group. All subjects underwent urine analysis, complete blood picture, liver function tests, kidney function tests, albumin/ creatinine ratio, antinuclear antibody, anti-double stranded DNA (anti-dsDNA) antibody, C3, C4, and anti-C1q Ab. All patients in the first group underwent renal biopsy and pathological diagnosis showed: Class II in two patients, Class III and IV in nine patients, and Class V in four patients. Anti-C1q Ab were found in the serum of SLE patients and not in the control group and showed an association with active lupus with much higher concentration in active renal group, specifically those with severe renal histological lesions (proliferative form). There was significant statistical positive correlation between anti-C1q Ab with anti-dsDNA and SLE disease activity index in both active groups. There was statistically significant negative correlation between anti-C1q Ab with C3 and C4 in both active groups. Anti-C1q Ab could be used as useful marker for active lupus, especially with nephritis.

High Prevalence and Disease Correlation of Autoantibodies Against p40 Encoded by Long Interspersed Nuclear Elements in Systemic Lupus Erythematosus.

Long interspersed nuclear element 1 (LINE-1) encodes 2 proteins, the RNA binding protein p40 and endonuclease and reverse transcriptase (open-reading frame 2p [ORF2p]), which are both required for LINE-1 to retrotranspose. In cells expressing LINE-1, these proteins assemble with LINE-1 RNA and additional RNA binding proteins, some of which are well-known autoantigens in patients with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether SLE patients also produce autoantibodies against LINE-1 p40. Highly purified p40 protein was used to quantitate IgG autoantibodies in serum from 172 SLE patients and from disease controls and age-matched healthy subjects by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Preparations of p40 that also contained associated proteins were analyzed by immunoblotting with patient sera. Antibodies reactive with p40 were detected in the majority of patients and many healthy controls. Their levels were higher in patients with SLE, but not those with systemic sclerosis, compared to healthy subjects (P = 0.01). Anti-p40 reactivity was higher in patients during a flare than in patients with disease in remission (P = 0.03); correlated with the SLE Disease Activity Index score (P = 0.0002), type I interferon score (P = 0.006), decrease in complement C3 level (P = 0.0001), the presence of anti-DNA antibodies (P < 0.0001) and anti-C1q antibodies (P = 0.004), and current or past history of nephritis (P = 0.02 and P = 0.003, respectively); and correlated inversely with age (r = -0.49, P < 0.0001). SLE patient sera also reacted with p40-associated proteins. Autoantibodies reacting with LINE-1 p40 characterize a population of SLE patients with severe and active disease. These autoantibodies may represent an early immune response against LINE-1 p40 that does not yet by itself imply clinically significant autoimmunity, but may represent an early, and still reversible, step toward SLE pathogenesis.

Lupus serum IgG induces microglia activation through Fc fragment dependent way and modulated by B-cell activating factor

BackgroundNeuropsychiatric manifestations are frequent in patients with systemic lupus erythematosus (SLE), yet the etiology and pathogenesis of brain damage in SLE remains unclear. Because the production of autoantibodies, formation and deposition of immunocomplexes are major serological characteristics of SLE, the elevated level of serum immunoglobulin may contribute to brain tissue injury of SLE. To testify this, in this study, we examined whether immunoglobulin G (IgG) in the serum of SLE patients affects the cellular functions in central nervous system and the potential mechanism.MethodsIn vivo intracerebral injection of SLE-serum in mouse was used to activate microglia and the production of pro-inflammatory cytokine was assessed by ELISA. Sera was divided into IgG and IgG depleted fractions, while IgG was further divided into Fc and Fab fragments to examine which part has an effect on microglia. Flow cytometry, immunofluorescence and quantitative PCR (qPCR) were used to verify the synergistic effect of B-cell activating factor (BAFF) on IgG stimulation of microglia.ResultsWe found that IgG in lupus sera can induce M1 activation of brain microglia following intraventricular injection into normal mice, and BAFF facilitates this process. In vitro, we identified that IgG bound to microglia through Fc rather than Fab fragments, and BAFF up-regulated the expression of Fc receptors (FcγR) on the surface of microglia, consequently, promote IgG binding to microglia.ConclusionOur results suggest that lupus serum IgG causes inflammatory responses of microglia by involving the Fc signaling pathway and the activity could be up-regulated by BAFF. Accordingly, disruption of the FcγR-mediated signaling pathway and blockade of microglia activation may be a therapeutic target in patients with neuropsychiatric lupus erythematosus.

Neutrophil extracellular traps-associated markers are elevated in patients with systemic lupus erythematosus.

Neutrophil extracellular traps (NETs) are the main source of autoantigens in systemic lupus erythematosus (SLE). The aim of this study was to evaluate the clinical importance of NETs-associated markers in SLE. We compared NETs-associated markers in SLE patients (n = 111) with healthy controls (n = 50). Moreover, in 35 patients with drug-naïve SLE (n = 35), we investigated correlation between NETs-associated markers [DNase I concentration, myeloperoxidase (MPO) activity, anti-MPO antibodies, cell-free DNA (cfDNA), NETolytic activity] with serological parameters [anti-dsDNA antibodies, C3, C4 and B-cell activating factor (BAFF) levels] and disease activity measured by modified SLE Disease Activity Index (M-SLEDAI-2K). In comparison with healthy controls, SLE patients had higher cfDNA, MPO activity, anti-MPO antibodies (p < 0.001), BAFF and DNase I concentration (p < 0.01). Contrary, NETolytic activity was lower in SLE patients (p < 0.05), despite higher concentration of DNase I. MPO activity and cfDNA levels showed correlation with DNase I concentration (p < 0.001, p < 0.01, respectively). BAFF levels correlated with cfDNA, DNase I concentration and MPO activity (p < 0.05). Anti-dsDNA antibodies showed correlation with MPO activity (p < 0.01), cfDNA and BAFF levels (p < 0.001). Anti-dsDNA and C3 levels were independent predictors of M-SLEDAI-2K in multivariate analysis (p < 0.01). We demonstrated that sera of SLE patients have decreased NETolytic activity, leading to increased levels of various NETs-associated markers, which correlate with anti-dsDNA antibodies in drug-naïve SLE. We showed that BAFF participates in a complex relationship between NETosis and anti-dsDNA antibodies production. These findings have important implications for a better understanding of SLE pathogenesis and development of therapy that inhibits NETs persistence and disease progression.

Novel Autoantibodies Related to Cell Death and DNA Repair Pathways in Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune syndrome characterized by various co-existing autoantibodies (autoAbs) in patients’ blood. However, the full spectrum of autoAbs in SLE has not been comprehensively elucidated. In this study, a commercial platform bearing 9400 antigens (ProtoArray) was used to identify autoAbs that were significantly elevated in the sera of SLE patients. By comparing the autoAb profiles of SLE patients with those of healthy controls, we identified 437 IgG and 1213 IgM autoAbs that the expression levels were significantly increased in SLE (P < 0.05). Use of the ProtoArray platform uncovered over 300 novel autoAbs targeting a broad range of nuclear, cytoplasmic, and membrane antigens. Molecular interaction network analysis revealed that the antigens targeted by the autoAbs were most significantly enriched in cell death, cell cycle, and DNA repair pathways. A group of autoAbs associated with cell apoptosis and DNA repair function, including those targeting APEX1, AURKA, POLB, AGO1, HMGB1, IFIT5, MAPKAPK3, PADI4, RGS3, SRP19, UBE2S, and VRK1, were further validated by ELISA and Western blot in a larger cohort. In addition, the levels of autoAbs against APEX1, HMGB1, VRK1, AURKA, PADI4, and SRP19 were positively correlated with the level of anti-dsDNA in SLE patients. Comprehensive autoAb screening has identified novel autoAbs, which may shed light on potential pathogenic pathways leading to lupus.

MMP-9/Gelatinase B Degrades Immune Complexes in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) is a common and devastating autoimmune disease, characterized by a dysregulated adaptive immune response against intracellular antigens, which involves both autoreactive T and B cells. In SLE, mainly intracellular autoantigens generate autoantibodies and these assemble into immune complexes and activate the classical pathway of the complement system enhancing inflammation. Matrix metalloproteinase-9 (MMP-9) levels have been investigated in the serum of SLE patients and in control subjects. On the basis of specific studies, it has been suggested to treat SLE patients with MMP inhibitors. However, some of these inhibitors induce SLE. Analysis of LPR−/−MMP-9−/− double knockout mice suggested that MMP-9 plays a protective role in autoantigen clearance in SLE, but the effects of MMP-9 on immune complexes remained elusive. Therefore, we studied the role of MMP-9 in the clearance of autoantigens, autoantibodies and immune complexes and demonstrated that the lack of MMP-9 increased the levels of immune complexes in plasma and local complement activation in spleen and kidney in the LPR−/− mouse model of SLE. In addition, we showed that MMP-9 dissolved immune complexes from plasma of lupus-prone LPR−/−/MMP-9−/− mice and from blood samples of SLE patients. Surprisingly, autoantigens incorporated into immune complexes, but not immunoglobulin heavy or light chains, were cleaved by MMP-9. We discovered Apolipoprotein-B 100 as a new substrate of MMP-9 by analyzing the degradation of immune complexes from human plasma samples. These data are relevant to understand lupus immunopathology and side-effects observed with the use of known drugs. Moreover, we caution against the use of MMP inhibitors for the treatment of SLE.

16 α-Hydroxyestrone induced adduct generate high affinity autoantibodies in SLE

PurposeIncreased 16 α-hydroxyestrone (16 α-OHE1) and autoantibodies against histone H1 (H1) have been well described in systemic lupus erythematosus (SLE), but the combination effects of 16 α-OHE1 and H1 remains unclear. Here, we tried to assess the affinity and binding specificity of SLE autoantibodies against the 16 α-OHE1-H1 adduct. IgG was induced against this adduct and was also used as immunochemical probe for the estimation of 16 α-OHE1 in the serum of SLE patients. Materials and methodsThe affinity and binding specificities of SLE autoantibodies against 16 α-OHE1-H1 were determined by direct binding and inhibition ELISA as well as quantitative precipitation titration in 60 patients and 30 control subjects. ResultsPurified SLE autoantibodies showed greater recognition to 16 α-OHE1-H1 over H1 (p < 0.05) or 16 α-OHE1 (p < 0.001). The relative affinity of SLE autoantibodies for 16 α-OHE1-H1, H1 and 16 α-OHE1 was 1.41 × 10−7, 1.31 × 10−6, and 1.03 × 10−6, respectively. The concentration of 16 α-OHE1 in the sera of SLE patients was significantly higher than controls (p < 0.05) as estimated by anti-16 α-OHE1-H1 antibodies. ConclusionsHigh affinity of 16 α-OHE1-H1 with SLE autoantibodies might suggest an antigenic role of this adduct in the production of these autoantibodies. The anti-16 α-OHE1-H1 antibody is a good immunochemical probe to measure 16 α-OHE1 in different SLE sera.

Diagnostic potential of metastasis-associated-lung-adenocarcinoma-transcript-1 (MALAT-1) and TNFα and hnRNPL related immunoregulatory long non-coding RNA (THRIL) in systemic lupus erythematosus patients: Relation to disease activity

Aim of the workTo determine expression levels and diagnostic value of metastasis-associated-lung-adenocarcinoma-transcript-1 (MALAT-1) and TNFα and hnRNPL related immunoregulatory long non-coding RNA (THRIL) in systemic lupus erythematosus (SLE), and to assess their role in the clinical characteristics of SLE and disease activity. Patients and methodsStudy included 40 patients with SLE and 30 matched controls. SLE Disease Activity Index (SLEDAI) score was assessed. Expression levels of MALAT-1 and THRIL were detected in the serum by using Real-time polymerase chain reaction and 2−ΔΔCT method. ResultsMean age of patients was 40.1 ± 9 years (25–55 years), they were 38 females and 2 males and disease duration was 16.5 ± 3.9 years. Their mean SLEDAI was 5.8 ± 5.3. Expression levels of MALAT-1 and THRIL were found to be significantly upregulated in the serum of SLE patients compared with controls (set as 1). MALAT-1 fold change = 3.7 ± 3.8 (p = 0.009), and THRIL fold change = 3.6 ± 3.4 (p = 0.026). There were significant correlations between MALAT-1 with THRILL (r = 0.44, p = 0.005), proteinuria (r = 0.45, p = 0.006), erythrocyte sedimentation rate (r = 0.43, p = 0.006) and SLEDAI (r = 0.36, p = 0.024). No significant correlations were found between THRIL and study parameters. Sensitivity and specificity of MALAT-1 and THRIL were determined (sensitivity 67.5% and 65% respectively), (specificity 100% for both, total accuracy 80% and 81.4% respectively), and the combined effect of both increased sensitivity and total accuracy to 70% and 82.9% respectively. THRIL was a significant predictor for SLE disease (p = 0.02). ConclusionMALAT-1 and THRIL may be potential diagnostic biomarkers for SLE and only MALAT-1 may be valuable in detecting disease activity.

Association of Serum T cell Immunoglobulin Domain and Mucin-3 and Interleukin-17 with Systemic Lupus Erythematosus.

BackgroundPrevious studies have shown that T cell immunoglobulin domain and mucin-3 (Tim-3) and interleukin-17 (IL-17) are implicated in the development of several autoimmune diseases. However, it is unclear whether these proteins contribute to the pathogenesis of systemic lupus erythematosus (SLE). The purpose of this study was to evaluate SLE patient serum Tim-3 and IL-17 levels, and to assess correlations between these proteins and major clinical parameters of SLE.Material/MethodsOverall, 55 SLE patients and 55 healthy controls were recruited in a case-control study. Serum Tim-3 and IL-17 levels were quantified using an enzyme-linked immunosorbent assay (ELISA) kit.ResultsSerum Tim-3 and IL-17 levels in SLE patients were significantly elevated relative to healthy controls (all P<0.05). Serum Tim-3 levels were significantly lower in SLE patients with nephritis than in those SLE without nephritis (P<0.05), while no statistically significant correlation between serum IL-17 and nephritis was detected (P>0.05). Serum Tim-3 with IL-17 levels were positively correlated in SLE patients (rs=0.817, P<0.01); however, no statistically significant correlation was found between serum Tim-3 or IL-17 levels and systemic lupus erythematosus disease activity index (SLEDAI) scores in those with SLE (all P>0.05). In addition, serum Tim-3 was associated with central lesions in SLE patients, while there were no significant correlations between serum Tim-3 or IL-17 levels and other SLE clinical parameters.ConclusionsIncreased serum Tim-3 and IL-17 levels and their clinical associations in SLE patients suggest their possible role in this disease.

Shotgun Lipidomics Revealed Altered Profiles of Serum Lipids in Systemic Lupus Erythematosus Closely Associated with Disease Activity

The pathogenesis of systemic lupus erythematosus (SLE) remains elusive. It appears that serum lipid metabolism is aberrant in SLE patients. Determination of lipid profiles in the serum of SLE patients may provide insights into the underlying mechanism(s) leading to SLE and may discover potential biomarkers for early diagnosis of SLE. This study aimed to identify and quantify the profile of serum lipids in SLE patients (N = 30) with our powerful multi-dimensional mass spectrometry-based shotgun lipidomics platform. Multivariate analysis in the form of partial least squares-discriminate analysis was performed, and the associations between the changed lipids with cytokines and SLE disease activity index (SLEDAI) were analyzed using a multiple regression method. The results of this study indicated that the composition of lipid species including diacyl phosphatidylethanolamine (dPE) (16:0/18:2, 18:0/18:2, 16:0/22:6, 18:0/20:4, and 18:0/22:6), 18:2 lysoPC (LPC), and ceramide (N22:0 and N24:1) was significantly altered in SLE patients with p < 0.05 and variable importance of the projection (VIP) > 1 in partial least squares-discriminate analysis (PLS-DA). There existed significant associations between IL-10, and both 18:0/18:2 and 16:0/22:6 dPE species with p < 0.0001 and predicting 85.7 and 95.8% of the variability of IL-10 levels, respectively. All the altered lipid species could obviously predict IL-10 levels with F (8, 21) = 3.729, p = 0.007, and R2 = 0.766. There was also a significant correlation between the SLEDAI score and 18:0/18:2 dPE (p = 0.031) with explaining 22.6% of the variability of SLEDAI score. Therefore, the panel of changed compositions of dPE and ceramide species may serve as additional biomarkers for early diagnosis and/or prognosis of SLE.

Effect of storage duration on cytokine stability in human serum and plasma

Quantification of analytes such as cytokines in serum samples is intrinsic to translational research in immune diseases. Optimising pre-analytical conditions is critical for ensuring study quality, including evaluation of cytokine stability. We aimed to evaluate the effect on cytokine stability of storage duration prior to freezing of serum, and compare to plasma samples obtained from patients with systemic lupus erythematosus (SLE). Protein stability was analysed by simultaneously quantifying 18 analytes using a custom multi-analyte profile in SLE patient serum and plasma samples that had been prospectively stored at 4 °C for pre-determined periods between 0 and 30 days, prior to freezing. Six analytes were excluded from analysis, because most tested samples were above or below the limit of detection. Amongst the 12 analysed proteins, 11 did not show significant signal degradation. Significant signal degradation was observed from the fourth day of storage for a single analyte, CCL19. Proteins levels were more stable in unseparated serum compared to plasma for most analytes, with the exception of IL-37 which appears slightly more stable in plasma. Based on this, a maximum 3 days of storage at 4 °C for unseparated serum samples is recommended for biobanked samples intended for cytokine analysis in studies of human immune disease.

Periodontal pathogen exposure facilitates disease activity in Systemic Lupus Erythematosus

Abstract This study was undertaken to evaluate whether exposure to subgingival plaque bacteria and periodontal dysbiosis influences disease parameters in systemic lupus erythematosus (SLE). Circulating antibodies to periodontal bacteria are surrogate markers to determine an ongoing bacterial burden, or are indicators of past exposure. SLE patient sera in the Oklahoma Lupus Cohort (n=303) were used to measure antibody titers against periodontal pathogens (A. actinomycetemcomitans, P. gingivalis, and T. denticola) and commensals (C. ochracea and S. gordonii) by ELISA. Correlations between anti-bacterial antibodies and clinical parameters of SLE including autoantibodies, complement, and disease activity (SLEDAI and BILAG) were studied. The effect of oral infection with A. actinomycetemcomitans on SLE in NZM2328 lupus mice was evaluated. SLE patients had varying amounts of antibodies to different oral bacteria. The antibody titers against A. actinomycetemcomitans, P. gingivalis, T. denticola, and C. ochracea were higher in patients positive for anti-dsDNA, and in patients with low complement. Only antibodies to A. actinomycetemcomitans and P. gingivalis, but not T. denticola, were associated with higher disease activity. A. actinomycetemcomitans infected NZM2328 mice developed an increase in histone reactive T cells and an accelerated onset of proteinuria, and lupus nephritis. Our results indicate that exposure to only specific pathogenic periodontal bacteria influences disease activity in SLE patients. The results from lupus mice suggest that this may be due to the amplification of local autoimmunity in response to infection. These findings provide a rationale for assessing and improving periodontal health in SLE patients.