Increased antibody levels to stage-specific Epstein-Barr virus antigens in systemic autoimmune diseases reveal a common pathology.

The immune responses to antigens from different stages of the Epstein-Barr virus (EBV) life cycle were investigated in systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), and systemic sclerosis (SSc) to gain knowledge of EBV's involvement in the etiology of systemic autoimmune diseases (SADs) and for an overview of the humoral immune responses against EBV. Investigations were performed by the use of ELISA. IgM, IgA, and IgG antibody binding to 11 EBV antigens: EBNA1, EBNA2, BALF5, EAD, BALF2, EA/R, VCA p18, VCA p23, gB, gp350, and gp42 were examined in serum pools from SAD patients and healthy controls (HCs). Increased antibody levels against the 11 EBV antigens in the SAD pools were seen compared to the HC pool. Specifically, SLE was characterized by strongly increased IgA to EAD both compared to HCs and other SADs, and RA was characterized by increased IgM levels to several EBV antigens. The SADs may be partly distinguished by their differential immune responses to various antigens in the EBV life cycle. All together, these findings support an association between EBV infection and SADs.

Development of a Neo-Epitope Specific Assay for Serological Assessment of Type VII Collagen Turnover and Its Relevance in Fibroproliferative Disorders.

Type VII collagen is the main component of the anchoring fibrils connecting the basement membrane to the underlying interstitial matrix. Mutations in the type VII collagen gene cause dystrophic epidermolysis bullosa. Increased levels of type VII collagen in the skin have been reported in patients with systemic sclerosis (SSc), whereas reduced levels in the airways have been related to asthma. This indicates that type VII collagen plays an important part in upholding tissue integrity and that its remodeling may lead to pathological states. The aim of this study was to investigate the role of type VII collagen remodeling in fibroproliferative disorders. We produced monoclonal antibody targeting a specific fragment of type VII collagen (C7M) released to the systemic circulation and developed a neo-epitope specific competitive enzyme-linked immunosorbent assay (ELISA). Biological relevance was evaluated in serum from patients with SSc or chronic obstructive pulmonary disease (COPD). The C7M ELISA was technically robust and specific for the C7M neo-epitope. Serum C7M levels were significantly elevated in two cohorts of patients with SSc and in patients with COPD as compared with healthy individuals (P < 0.0001). The C7M ELISA enabled quantification of type VII collagen turnover in serum. Elevated serum C7M levels indicated that the turnover rate of type VII collagen was significantly increased in patients with SSc or COPD, suggesting a pathological role. Thus, the C7M ELISA may become useful in future investigations of type VII collagen turnover in fibroproliferative disorders, and it may prove a valuable tool for evaluating novel anti-fibrotic drugs.

Distinct proteome pathology of circulating microparticles in systemic lupus erythematosus.

BACKGROUND: The pathogenesis of systemic lupus erythematosus (SLE) is poorly understood but has been linked to defective clearance of subcellular particulate material from the circulation. This study investigates the origin, formation, and specificity of circulating microparticles (MPs) in patients with SLE based on comprehensive MP proteome profiling using patients with systemic sclerosis (SSc) and healthy donors (HC) as controls. METHODS: We purified MPs from platelet-poor plasma using differential centrifugation of samples from SLE (n = 45), SSc (n = 38), and two sets of HC (n = 35, n = 25). MP proteins were identified and quantitated after trypsin digestion by liquid chromatography-tandem mass spectrometry. The abundance of specific proteins was compared between the groups using univariate statistics and false discovery rate correction for multiple comparisons. Specific proteins and protein ratios were explored for diagnostic and disease activity information using receiver-operating characteristic curves and by analysis of correlations of protein abundance with disease activity scores. RESULTS: We identify and quantitate more than 1000 MP proteins and show that a subpopulation of SLE-MPs (which we propose to call luposomes) are highly specific for SLE, i.e. not found in MP preparations from HC or patients with another autoimmune, systemic disease, SSc. In SLE-MPs platelet proteins and mitochondrial proteins are significantly diminished, cytoskeletal proteins deranged, and glycolytic enzymes and apoptotic proteins significantly increased. CONCLUSIONS: Normal MPs are efficiently removed in SLE, but aberrant MPs, derived from non-lymphoid leukocytes, are less efficiently removed and abundantly produced leading to an altered MP proteome in SLE. The data suggest that an abnormal generation of MPs may partake in the pathology of SLE and that new diagnostic, monitoring, and treatment strategies targeting these processes may be advantageous.

Efficient evaluation of humoral immune responses by the use of serum pools.

BACKGROUND: Collection and testing of individual serum samples are often used in research to gain knowledge about e.g. the humoral response against bacteria or virus. This is a valid but time-consuming method and might be a waste of valuable serum samples for inefficient research. So far, no study has considered using serum pools as a quick and efficient screening method to confirm or deny hypotheses. METHODS: We created serum pools from four different patient groups (systemic lupus erythematosus n=85, rheumatoid arthritis n=77, Sjögren's syndrome n=91, systemic sclerosis n=66) and one healthy control group (n=67). Each serum pool was analyzed using three well-known immunoassays: enzyme-linked immunosorbent assay (ELISA), line blot, and immunofluorescence microscopy (anti-nuclear antibody (ANA) screening). The presence of Epstein-Barr virus (EBV) EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies was used to validate serum pools as an efficient tool for further investigations by comparison to previous findings in this area. RESULTS: The presence of EBV EA/D-, EBNA-1-, VCA p23-, and gp350-directed antibodies in each pool was consistent within the obtained ELISA and line blot results, as increased titers of IgG against the four antigens were found in all patient serum pools and also in individual sera regarding gp350. These results correspond to previous findings on individual samples from patients with these diseases. The presence of ANAs was observed in all four patient serum pools and not in the HC pool by both line blots and immunofluorescence microscopy, which corresponds with the expectations and further corroborate the application of serum pools for screenings. CONCLUSION: We developed and validated the use of serum pools that reliably and rapidly can confirm or deny hypotheses, which enables a more efficient research concentrating on the most evident factors.

Cross-sectional study of soluble selectins, fractions of circulating microparticles and their relationship to lung and skin involvement in systemic sclerosis.

BACKGROUND: Endothelial damage and activation may play central roles in the pathogenesis of systemic sclerosis (SSc) and are reflected by microparticles (MPs) and soluble selectins. The objective of this study was to determine if these potential biomarkers are associated with specific organ involvements or cutaneous subgroups of SSc patients. METHOD: MPs in platelet-poor plasma from 121 patients with SSc, 79 and 42 with limited and diffuse cutaneous disease, respectively, were characterized by flow cytometry for their capacity to bind annexin V in combination with surface markers of either platelets (PMPs), leukocytes (LMPs) or endothelial cells (EMPs). Soluble E- and P-selectin levels were determined in plasma. By correlation analyses, this was held against involvement of skin, lung function, lung fibrosis, pulmonary artery hypertension, and serology. RESULTS: None of the markers were associated with cutaneous subgroups of SSc. Concentrations of annexin V non-binding EMPs and annexin V non-binding LMPs were negatively correlated to pulmonary diffusing capacity (DLCO) (r = -0.28; p = 0.003; r = -0.26; p = 0.005) and forced vital capacity (FVC) (r = -0.24; p = 0.009; r = -0.29; p = 0.002), driven by patients with limited and diffuse cutaneous disease, respectively. Soluble E-selectin levels correlated negatively to DL(CO) (r = -0.21, p = 0.03) and FVC (r = -0.25; p = 0.007); and soluble P-selectin correlated negatively to DL(CO) (r = -0.23, p = 0.01). CONCLUSION: Negative correlations between annexin V non-binding EMP and LMP concentrations with lung function parameters (DL(CO) and FVC) differed between limited and diffuse cutaneous subsets of SSc, indicative of various pathogeneses of lung involvement in SSc, possibly with a differential role of MPs.

The circulating cell-free microRNA profile in systemic sclerosis is distinct from both healthy controls and systemic lupus erythematosus.

OBJECTIVE: To evaluate the expression profile of cell-free circulating microRNA (miRNA) in systemic sclerosis (SSc), healthy controls (HC), and systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma and 45 different, mature miRNA were measured using quantitative PCR assays after reverse transcription. Samples (n = 189) were from patients with SSc (n = 120), SLE (n = 29), and from HC (n = 40). Expression data were clustered by principal components analysis, and diagnostically specific miRNA profiles were developed by leave-one-out cross-validation. Diagnostic probability scores were derived from stepwise logistic regression. RESULTS: Thirty-seven miRNA specificities were consistently detected and 26 of these were unaffected by SSc sample age and present in more than two-thirds of SSc samples. SSc cases showed a distinct expression profile with 14/26 miRNA significantly decreased (false discovery rate < 0.05) and 5/26 increased compared with HC. A 21-miRNA classifier gave optimum accuracy (80%) for discriminating SSc from both HC and SLE. The discrimination between HC and SSc (95% accuracy) was strongly driven by miRNA of the 17 ∼ 92 cluster and by miR-16, -223, and -638, while SLE and SSc differed mainly in the expression of miR-142-3p, -150, -223, and -638. Except for a weak correlation between anti-Scl-70 and miR-638 (p = 0.048), there were no correlations with other patient variables. CONCLUSION: Circulating miRNA profiles are characteristic for SSc compared with both HC and SLE cases. Some of the predicted targets of the differentially regulated miRNA are of relevance for transforming growth factor-ß signaling and fibrosis, but need to be validated in independent studies.

Plasma levels of galectin-3-binding protein reflect type I interferon activity and are increased in patients with systemic lupus erythematosus.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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). CONCLUSIONS: 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.

Unique protein signature of circulating microparticles in systemic lupus erythematosus.

OBJECTIVE: To characterize the unique qualities of proteins associated with circulating subcellular material in systemic lupus erythematosus (SLE) patients compared with healthy controls and patients with other chronic autoimmune diseases. METHODS: Using differential centrifugation and high-sensitivity nano-liquid chromatography tandem mass spectrometry, we systematically profiled proteins of microparticles (MPs) from SLE patients (n=12), systemic sclerosis (SSc) patients (n=6), and rheumatoid arthritis (RA) patients (n=6), as well as healthy controls (n=12). RESULTS: We identified 531 unique proteins and showed that the differences between healthy controls and patients with SLE with regard to the abundance of 248 proteins were highly statistically significant. Almost half of the proteins that were increased by >2-fold were complement proteins and Ig (increased by 100-4,000 times). MP Ig and complement loads also distinguished SLE from RA and SSc and correlated strongly with clinical SLE severity. Subsets of microtubule proteins, fibronectin, 14-3-3η, and desmosomal proteins as well as ficolin 2 and galectin 3 binding protein were also highly increased. In SLE MPs, levels of cytoskeletal, mitochondrial, and organelle proteins, including lysosome-associated membrane protein 1 and transforming growth factor ß1, were decreased. CONCLUSION: The data show that SLE patients have increased numbers of MPs that are heavily tagged for removal and fewer MPs with normal protein composition. SLE MPs are unique and specific proteins that represent novel leads for our understanding of SLE and for the development of new treatments of the disease.

A heparin-based method for flow cytometric analysis of microparticles directly from platelet-poor plasma in calcium containing buffer.

BACKGROUND: Characterization of circulating microparticles (MPs) is usually performed by flow cytometry. Annexin V, a protein that Ca(2+)-dependently binds to phosphatidylserine, has been used to define entire microparticle (MP) populations, but not all MPs bind AnxV. Recent reports have correlated AnxV negative MPs to clinical parameters in systemic diseases, which emphasize the importance of including characterization of AnxV-binding. An obstacle in flow cytometric analysis of AnxV-binding to MPs is that plasma may clot when adding the Ca(2+)-containing buffers. We here devise a simple method for comprehensive assessment of circulating MPs directly from platelet-poor plasma with characterization of AnxV-binding and of cellular origin of MPs. MATERIALS AND METHODS: With 49 samples (20 healthy controls and 29 SLE patients) a flow cytometric method analyzing MPs directly from platelet-poor plasma was developed and compared with an established, more laborious method. The method relies on using heparin to inhibit plasma coagulation induced by Ca(2+) and subsequent incubation with labeling reagents directly in platelet-poor plasma. RESULTS: In comparison with standard methods the new direct method showed low variability (1-12% in total MP measurement), had higher MP counts (i.e. prevents loss of MPs), was less time consuming (saved 2/3 of sample processing time) and results correlated well with an established method of analysis of washed MPs by flow cytometry (r>0.7; p<0.0001). Additionally heparin was shown not to impact MP counts, longtime storage did not alter MP concentrations, and distinct effects of freezing on MP characteristics were confirmed. CONCLUSIONS: The direct method reduces variability due its simplicity and faster handling, and it saves sample material. It is a convenient, fast, and reproducible method for assessing the population of circulating MPs and correlates well with more cumbersome approaches. These benefits make the method well suited for large studies.

Quantitative proteome profiling of normal human circulating microparticles.

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated. Of these, 334 (63%) were present in all samples and represent an MP core proteome. Technical triplicates showed <10% variation in intensity within a dynamic range of almost 5 decades. Differences due to variable MP numbers and losses during preparative steps could be normalized using cytoskeletal MP protein intensities. Our results establish a reproducible LC-MS/MS procedure, provide a simple and robust MP preparation method, and yield a baseline MP proteome for future studies of MPs in health and disease.

Increased IgG on cell-derived plasma microparticles in systemic lupus erythematosus is associated with autoantibodies and complement activation.

OBJECTIVE: To quantify immunoglobulin and C1q on circulating cell-derived microparticles (MPs) in patients with systemic lupus erythematosus (SLE) and to determine whether immunoglobulin and C1q levels are correlated with clinical and serologic parameters. METHODS: Sixty-eight clinically well-characterized SLE patients, 38 healthy controls, 6 patients with systemic sclerosis (SSc), and 6 patients with rheumatoid arthritis (RA) were included. The numbers of annexin V-binding MPs displaying IgG, IgM, or C1q were enumerated by flow cytometry. MP protein levels were determined by mass spectrometry in clinically defined subsets of SLE patients and controls. The MP IgG load was determined by flow cytometric analysis of all samples from SLE patients and healthy controls. RESULTS: SLE patients had significantly increased total and relative numbers of IgG-positive MPs (P = 0.0004), with a much higher average IgG load per MP (P < 0.0001) than healthy controls. Quantitative mass spectrometry of purified MPs verified significantly increased IgG, IgM, and C1q levels in SLE patients. In RA and SSc patients, the average IgG load per MP was significantly lower than in SLE patients (P = 0.006 and P = 0.05, respectively). Also, the IgM load and C1q load per MP were significantly higher in SLE patients than in the control groups (P < 0.05), except for IgM in the RA group. IgG-positive MPs were significantly associated with the presence of anti-double-stranded DNA, anti-extractable nuclear antigen, and antihistone antibodies, with total IgG, and with decreased leukocyte counts. Average IgG load per MP was associated with lower concentrations of MPs, the presence of anti-C1q antibodies, and complement consumption. CONCLUSION: Our findings indicate that circulating cell-derived MPs in SLE patients carry increased loads of IgG, IgM, and C1q and that IgG MPs are associated with autoantibodies and complement activation. The findings link immunologic reactions on MPs with the etiology of SLE.