Circulating plasma microRNAs in systemic sclerosis-associated pulmonary arterial hypertension.

OBJECTIVES: SSc-associated pulmonary arterial hypertension (SSc-APAH) is a late but devastating complication of SSc. Early identification of SSc-APAH may improve survival. We examined the role of circulating miRNAs in SSc-APAH. METHODS: Using quantitative RT-PCR the abundance of mature miRNAs in plasma was determined in 85 female patients with ACA-positive lcSSc. Twenty-two of the patients had SSc-APAH. Sixty-three SSc controls without PAH were matched for disease duration. Forty-six selected miRNA plasma levels were correlated with clinical data. Longitudinal samples were analysed from 14 SSc-APAH and 27 SSc patients. RESULTS: The disease duration was 12 years for the SSc-APAH patients and 12.7 years for the SSc controls. Plasma expression levels of 11 miRNAs were lower in patients with SSc-APAH. Four miRNAs displayed higher plasma levels in SSc-APAH patients compared with SSc controls. There was significant difference between groups for miR-20a-5p and miR-203a-3p when correcting for multiple comparisons (P = 0.002 for both). Receiver operating characteristics curve showed AUC = 0.69-0.83 for miR-21-5p and miR-20a-5p or their combination. miR-20a-5p and miR-203a-3p correlated inversely with NT-pro-Brain Natriuretic Protein levels (r = -0.42 and -0.47). Mixed effect model analysis could not identify any miRNAs as predictor of PAH development. However, miR-20a-5p plasma levels were lower in the longitudinal samples of SSc-APAH patients than in the SSc controls. CONCLUSIONS: Our study links expression levels of the circulating plasma miRNAs, especially miR-20a-5p and miR-203a-3p, to the occurrence of SSc-APAH in female patients with ACA-positive lcSSc.

The biomarker potential of cell-free microRNA from cerebrospinal fluid in Parkinsonian Syndromes.

BACKGROUND: MicroRNAs are small noncoding RNAs involved in the post-transcriptional regulation of protein synthesis. Extracellular microRNAs are accessible in a stable form in biofluids. OBJECTIVES: The aim was to identify individual microRNAs and/or subsets of microRNAs in CSF with biomarker potential and thus identify specific putative pathophysiological pathways. METHODS: In a two-step exploratory study design of PD, MSA, PSP, and controls, we initially profiled CSF microRNAs in a pilot cohort (n = 40) by screening for 372 microRNAs. Subsequently, we attempted to validate findings in an independent study cohort in CSF (n = 118) and ethylenediaminetetraacetic acid plasma (n = 114). This study cohort encompassed 46 microRNAs, of which 26 were singled out from the pilot cohort, and an additional 20 microRNAs were added based on previous publications. The most accurate diagnostic microRNA classifiers were identified in a multivariable logistic regression model adjusted for age and sex. RESULTS: A set of three microRNAs in CSF discriminated PD and MSA from controls with good diagnostic accuracy by receiver operating characteristics curve evaluation. The microRNAs were for PD versus controls: miR-7-5p, miR-331-5p, and miR-145-5p (area under the curve = 0.88) and MSA versus controls: miR-7-5p, miR-34c-3p, and miR-let-7b-5p (area under the curve = 0.87). The classifier that best distinguished MSA and PD consisted of two microRNAs: miR-9-3p and miR-106b-5p (area under the curve = 0.73). A single microRNA, miR-106b-5p, provided the best discrimination between PD and PSP (area under the curve = 0.85) in the CSF. CONCLUSIONS: Levels of specific trios of CSF-microRNAs discriminate well between α-synucleinopathies (PD and MSA) and controls. The results need to be validated in larger, independent cohorts. © 2018 International Parkinson and Movement Disorder Society.

Paucimannose-Rich N-glycosylation of Spatiotemporally Regulated Human Neutrophil Elastase Modulates Its Immune Functions.

Human neutrophil elastase (HNE) is an important N-glycosylated serine protease in the innate immune system, but the structure and immune-modulating functions of HNE N-glycosylation remain undescribed. Herein, LC-MS/MS-based glycan, glycopeptide and glycoprotein profiling were utilized to first determine the heterogeneous N-glycosylation of HNE purified from neutrophil lysates and then from isolated neutrophil granules of healthy individuals. The spatiotemporal expression of HNE during neutrophil activation and the biological importance of its N-glycosylation were also investigated using immunoblotting, cell surface capture, native MS, receptor interaction, protease inhibition, and bacteria growth assays. Site-specific HNE glycoprofiling demonstrated that unusual paucimannosidic N-glycans, particularly Manα1,6Manß1,4GlcNAcß1,4(Fucα1,6)GlcNAcß, predominantly occupied Asn124 and Asn173. The equally unusual core fucosylated monoantenna complex-type N-sialoglycans also decorated these two fully occupied sites. In contrast, the mostly unoccupied Asn88 carried nonfucosylated paucimannosidic N-glycans probably resulting from low glycosylation site solvent accessibility. Asn185 was not glycosylated. Subcellular- and site-specific glycoprofiling showed highly uniform N-glycosylation of HNE residing in distinct neutrophil compartments. Stimulation-induced cell surface mobilization demonstrated a spatiotemporal regulation, but not cell surface-specific glycosylation signatures, of HNE in activated human neutrophils. The three glycosylation sites of HNE were located distal to the active site indicating glycan functions other than interference with HNE enzyme activity. Functionally, the paucimannosidic HNE glycoforms displayed preferential binding to human mannose binding lectin compared with the HNE sialoglycoforms, suggesting a glycoform-dependent involvement of HNE in complement activation. The heavily N-glycosylated HNE protease inhibitor, α1-antitrypsin, displayed concentration-dependent complex formation and preferred glycoform-glycoform interactions with HNE. Finally, both enzymatically active HNE and isolated HNE N-glycans demonstrated low micromolar concentration-dependent growth inhibition of clinically-relevant Pseudomonas aeruginosa, suggesting some bacteriostatic activity is conferred by the HNE N-glycans. Taken together, these observations support that the unusual HNE N-glycosylation, here reported for the first time, is involved in modulating multiple immune functions central to inflammation and infection.

Changes in kynurenine pathway metabolism in Parkinson patients with L-DOPA-induced dyskinesia.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective drug in the symptomatic treatment of Parkinson's disease, but chronic use is associated with L-DOPA-induced dyskinesia in more than half the patients after 10 years of treatment. L-DOPA treatment may affect tryptophan metabolism via the kynurenine pathway. Altered levels of kynurenine metabolites can affect glutamatergic transmission and may play a role in the development of L-DOPA-induced dyskinesia. In this study, we assessed kynurenine metabolites in plasma and cerebrospinal fluid of Parkinson's disease patients and controls. Parkinson patients (n = 26) were clinically assessed for severity of motor symptoms (UPDRS) and L-DOPA-induced dyskinesia (UDysRS). Plasma and cerebrospinal fluid samples were collected after overnight fasting and 1-2 h after intake of L-DOPA or other anti-Parkinson medication. Metabolites were analyzed in plasma and cerebrospinal fluid of controls (n = 14), Parkinson patients receiving no L-DOPA (n = 8), patients treated with L-DOPA without dyskinesia (n = 8), and patients with L-DOPA-induced dyskinesia (n = 10) using liquid chromatography-mass spectrometry. We observed approximately fourfold increase in the 3-hydroxykynurenine/kynurenic acid ratio in plasma of Parkinson's patients with L-DOPA-induced dyskinesia. Anthranilic acid levels were decreased in plasma and cerebrospinal fluid of this patient group. 5-Hydroxytryptophan levels were twofold increased in all L-DOPA-treated Parkinson's patients. We conclude that a higher 3-hydroxykynurenine/kynurenic acid ratio in plasma may serve as a biomarker for L-DOPA-induced dyskinesia. Longitudinal studies including larger patients cohorts are needed to verify whether the changes observed here may serve as a prognostic marker for L-DOPA-induced dyskinesia.

Decreased platelet size is associated with platelet activation and anti-phospholipid syndrome in systemic lupus erythematosus.

Objectives: . SLE is an autoimmune disease with increased cardiovascular morbidity and platelet activation. In the general population, increased platelet size predicts platelet reactivity and cardiovascular disease. The aim of this study was to investigate whether platelet size related to platelet activation and cardiovascular disease in SLE. Methods: . Fresh blood samples from SLE patients ( n = 148), healthy volunteers ( n = 79) and disease controls ( n = 40) were analysed for platelet size and activation by flow cytometry, ELISA and cell count. Associations to manifest cardiovascular disease, venous thrombosis and APS were adjusted for traditional cardiovascular risk factors using logistic regression analysis. Results: . SLE patients had decreased platelet size as compared with healthy controls ( P = 0.003). In SLE, decreased platelet size was related to increased platelet activation, in particular microparticle formation ( P < 0.0001, r = -0.46) and release of serotonin from dense granules ( P < 0.001, r = 0.57). SLE patients with aCL had decreased platelet size ( P = 0.02) and aCL decreased platelet size in vitro ( P = 0.007). In contrast to the general population, increased platelet size was not associated with cardiovascular disease. Instead, decreased platelet size was associated with secondary APS, even after adjusting for traditional cardiovascular risk factors ( P = 0.01, odds ratio 3.58). Conclusion: . Platelet size is decreased in SLE patients and associated with microparticle formation and APS. Future studies are needed to determine the underlying mechanism(s) as well as the potential predictive value of small platelets for disease complications in SLE.

A review of studies of the proteomes of circulating microparticles: key roles for galectin-3-binding protein-expressing microparticles in vascular diseases and systemic lupus erythematosus.

Subcellular microvesicles (MVs) have attracted increasing interest during the past decades. While initially considered as inert cellular debris, several important roles for MVs in physiological homeostasis, cancer, cardiovascular, and autoimmune diseases have been uncovered. Although still poorly understood, MVs are involved in trafficking of information from cell-to-cell, and are implicated in the regulation of immunity, thrombosis, and coagulation. Different subtypes of extracellular MVs exist. This review focuses on the cell membrane-derived shedded MVs (ranging in size from 200 to 1000 nm) typically termed microparticles (MPs). The numbers and particularly the composition of MPs appear to reflect the state of their parental cells and MPs may therefore carry great potential as clinical biomarkers which can be elucidated and developed by proteomics in particular. Determination of the identity of the specific proteins and their quantities, i.e. the proteome, in complex samples such as MPs enables an in-depth characterization of the phenotypical changes of the MPs during disease states. At present, only a limited number of proteomic studies of circulating MPs have been carried out in healthy individuals and disease populations. Interestingly, these studies indicate that a small set of MP-proteins, in particular, overexpression of galectin-3-binding protein (G3BP) distinguish MPs in patients with venous thromboembolism and the systemic autoimmune disease, systemic lupus erythematosus (SLE). G3BP is important in cell-cell adhesion, clearance, and intercellular signaling. MPs overexpressing G3BP may thus be involved in thrombosis and hemostasis, vascular inflammation, and autoimmunity, further favoring G3BP as a marker of "pathogenic" MPs. MPs expressing G3BP may also hold a potential as biomarkers in other conditions such as cancer and chronic viral infections. This review highlights the methodology and results of the proteome studies behind these discoveries and places them in a pathophysiological and biomarker perspective.

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.

Amyloid-ß and α-Synuclein Decrease the Level of Metal-Catalyzed Reactive Oxygen Species by Radical Scavenging and Redox Silencing.

The formation of reactive oxygen species (ROS) is linked to the pathogenesis of neurodegenerative diseases. Here we have investigated the effect of soluble and aggregated amyloid-ß (Aß) and α-synuclein (αS), associated with Alzheimer's and Parkinson's diseases, respectively, on the Cu(2+)-catalyzed formation of ROS in vitro in the presence of a biological reductant. We find that the levels of ROS, and the rate by which ROS is generated, are significantly reduced when Cu(2+) is bound to Aß or αS, particularly when they are in their oligomeric or fibrillar forms. This effect is attributed to a combination of radical scavenging and redox silencing mechanisms. Our findings suggest that the increase in ROS associated with the accumulation of aggregated Aß or αS does not result from a particularly ROS-active form of these peptides, but rather from either a local increase of Cu(2+) and other ROS-active metal ions in the aggregates or as a downstream consequence of the formation of the pathological amyloid structures.

Flow-Induced Dispersion Analysis for Probing Anti-dsDNA Antibody Binding Heterogeneity in Systemic Lupus Erythematosus Patients: Toward a New Approach for Diagnosis and Patient Stratification.

Detection of immune responses is important in the diagnosis of many diseases. For example, the detection of circulating autoantibodies against double-stranded DNA (dsDNA) is used in the diagnosis of Systemic Lupus Erythematosus (SLE). It is, however, difficult to reach satisfactory sensitivity, specificity, and accuracy with established assays. Also, existing methodologies for quantification of autoantibodies are challenging to transfer to a point-of-care setting. Here we present the use of flow-induced dispersion analysis (FIDA) for rapid (minutes) measurement of autoantibodies against dsDNA. The assay is based on Taylor dispersion analysis (TDA) and is fully automated with the use of standard capillary electrophoresis (CE) based equipment employing fluorescence detection. It is robust toward matrix effects as demonstrated by the direct analysis of samples composed of up to 85% plasma derived from human blood samples, and it allows for flexible exchange of the DNA sequences used to probe for the autoantibodies. Plasma samples from SLE positive patients were analyzed using the new FIDA methodology as well as by standard indirect immunofluorescence and solid-phase immunoassays. Interestingly, the patient antibodies bound DNA sequences with different affinities, suggesting pronounced heterogeneity among autoantibodies produced in SLE. The FIDA based methodology is a new approach for autoantibody detection and holds promise for being used for patient stratification and monitoring of disease activity.

Specific autoantibody profiles and disease subgroups correlate with circulating micro-RNA in systemic sclerosis.

OBJECTIVE: To evaluate the expression profiles of cell-free plasma miRNAs in SSc and to characterize their correlation with disease subgroups (lcSSc and dcSSc) and with autoantibody profiles. METHODS: Using quantitative RT-PCR, the abundance of 45 mature miRNAs in plasma was determined in 95 patients (lcSSc = 63; dcSSc = 32), representing the following autoantibody subgroups: ACA, anti-DNA topoisomerase I, anti-RNA polymerase III and anti-U1-ribonucleoprotein. MiRNA data were correlated with clinical and paraclinical data. Multiple regression was used to model membership of the lcSSc, dcSSc and autoantibody subgroups, based on miRNA expression profiles. RESULTS: Thirty-six miRNAs were measurable in all samples. Four (miRNA-223, -181b, -342-3p and -184) were differently expressed in lcSSc and dcSSc (false discovery rate < 0.05). Ten miRNAs exhibited statistically significantly different levels in one or more autoantibody groups, and five (miRNA-409, -184, -92a, -29a and -101) remained significant after correction for multiple comparisons. Multiple regression models accurately predicted ACA and anti-DNA topoisomerase I antibody-positive patients (area under the curve (AUC) = 0.97 and 0.93, respectively) as well as membership of the dcSSc and lcSSc groups (AUC = 0.88). CONCLUSION: Circulating miRNA profiles differ between lcSSc and dcSSc patients and between patients with different autoantibodies. This is the first time autoantibody profiles, disease phenotypes and plasma miRNA profiles have been shown to correlate in an autoimmune disease. The data support a pathobiological role of miRNAs because specific miRNAs associate with autoantibody profiles of known diagnostic and prognostic value.

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 potential of microRNAs as biofluid markers of neurodegenerative diseases--a systematic review.

MicroRNAs (miRNA) are biological molecules transcribed from non-protein coding regions of the genome, participating in regulating cellular processes. MiRNAs in biofluids may possess neurodegenerative disease biomarker potential for screening tests, differential diagnosis and disease progression monitoring. This systematic review clarifies biomarker potential of miRNAs detected in biofluids of neurodegenerative disease patients. Thirty-three and ten miRNAs displayed significant expression between patients with multiple sclerosis and Alzheimer's disease, respectively, compared to healthy controls in minimum two studies. Thirty-eight miRNAs showed biomarker potential by distinguishing significantly between minimum two diseases. Summarized data directs future research towards discovering new biomarkers for neurodegenerative diseases.

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.

Circulating microRNA expression profiles associated with systemic lupus erythematosus.

OBJECTIVE: To evaluate the specificity of expression patterns of cell-free circulating microRNAs (miRNAs) in systemic lupus erythematosus (SLE). METHODS: Total RNA was purified from plasma, and 45 different specific, mature miRNAs were determined using quantitative reverse transcription-polymerase chain reaction assays. A total of 409 plasma samples were obtained from 364 different patients with SLE, healthy control subjects, and control subjects with other autoimmune diseases. The results in the primary cohort of 62 patients with SLE and 29 healthy control subjects were validated in 2 independent cohorts: a validation cohort comprising 68 patients with SLE and 68 healthy control subjects, and a disease control cohort comprising 20 patients with SLE (19 of whom were from the other validation cohort), 46 healthy control subjects, 38 patients with vasculitis, 18 patients with rheumatoid arthritis, and 20 immunosuppressed patients. RESULTS: Seven miRNAs were statistically significantly differentially expressed in plasma from patients with SLE. The expression of miRNA-142-3p (miR-142-3p) and miR-181a was increased, and the expression of miR-106a, miR-17, miR-20a, miR-203, and miR-92a was decreased. In addition, the expression of miR-342-3p, miR-223, and miR-20a was significantly decreased in SLE patients with active nephritis. A predictive model for SLE based on 2 or 4 miRNAs differentiated patients with SLE from control subjects (76% accuracy) when validated independently (P < 2 × 10(-9) ). Use of the 4-miRNA model provided highly significant differentiation between the SLE group and disease controls, except for those with vasculitis. CONCLUSION: Circulating miRNAs are systematically altered in SLE. A 4-miRNA signature was diagnostic of SLE, and a specific subset of miRNA profiles was associated with nephritis. All of the signature miRNAs target genes in the transforming growth factor ß signaling pathways. Other targets include regulation of apoptosis, cytokine-cytokine receptors, T cell development, and cytoskeletal organization. These findings highlight possible dysregulated pathways in SLE and suggest that circulating miRNA patterns distinguish SLE from other immunoinflammatory phenotypes.

MicroRNAs in liver disease.

MicroRNAs are small noncoding RNA molecules that regulate gene expression posttranscriptionally through complementary base pairing with thousands of messenger RNAs. They regulate diverse physiological, developmental, and pathophysiological processes. Recent studies have uncovered the contribution of microRNAs to the pathogenesis of many human diseases, including liver diseases. Moreover, microRNAs have been identified as biomarkers that can often be detected in the systemic circulation. We review the role of microRNAs in liver physiology and pathophysiology, focusing on viral hepatitis, liver fibrosis, and cancer. We also discuss microRNAs as diagnostic and prognostic markers and microRNA-based therapeutic approaches for liver disease.

Analysis of protein aggregation in neurodegenerative disease.

Pathological protein and peptide aggregation are key events in a number of chronic and devastating neurodegenerative conditions including dementias such as Alzheimer's and Creutzfeldt-Jakob's disease and other central nervous system diseases such as Parkinson's and Huntington's disease and amyotrophic lateral sclerosis. Analytical methods for studying protein aggregation in these diseases are important for mapping pathophysiological events and ultimately for the development of new therapies and better diagnostic tools.

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.

Characterization and stability of transthyretin isoforms in cerebrospinal fluid examined by immunoprecipitation and high-resolution mass spectrometry of intact protein.

Post-translational modifications (PTMs) contribute significantly to the complexity of proteins. PTMs may vary in certain patterns according to diseases and microenviroments making them potential markers for pathological processes. Human transthyretin (TTR) is a transporter of thyroxine and retinol in blood and cerebrospinal fluid (CSF). A single free cysteine thiol group in TTR possesses the ability to form mixed disulfides potentially related to diseases such as TTR amyloidosis and Alzheimer's disease (AD). Additionally, TTR-Cys10 S-thiolations might mirror the oxidative stress and redox balance of CSF. Here we describe a quick and gentle method for immunoprecipitating (IP) TTR from CSF with minimal introduction of sample-handling artifacts. A high-resolution mass spectrometer (LTQ-Orbitrap XL) was used in a simple setup with direct infusion that generates data suitable for confident assignment of TTR isoforms and validation of the protocol. Moreover, we demonstrate how simple storage of CSF at 4°C induces major oxidative modifications of TTR. Using the optimized method, we show data from a limited number of mild cognitive impairment (MCI) and AD patients. The protocol controls and minimizes the introduction of sample-handling artifacts during purification of TTR isoforms for high-resolution MS analysis.

ß(2)-microglobulin amyloidosis.

Dialysis-related amyloidosis (DRA) is a clinical syndrome of pain, loss of function and other symptoms due to the deposition of amyloid consisting of ß(2)-microglobulin (ß(2)m) in the musculoskeletal system. The condition is seen in patients who suffer from chronic kidney disease and are treated with hemodialysis for a long time. Even though ß(2)m easily can be manipulated to form amyloid in laboratory experiments under non-physiological conditions the precise mechanisms involved in the formation of ß(2)m-amyloid in patients with DRA have been difficult to unravel. The current knowledge which is reviewed here indicates that conformational fluctuations centered around the D-strand, the DE-loop, and around the cis-configured Pro32 peptide bond are involved in ß(2)m amyloidosis. Also required are highly increased concentrations of circulating ß(2)m and possibly various post-translational modifications mediated by the pro-inflammatory environment in uremic blood, together with the influence of divalent metal ions (specifically Cu(2 +)), uremic toxins, and dialysis-enhanced redox-processes. It seems plausible that domain-swapped ß(2)m dimers act as building blocks of ß-spine cross-ß -sheet fibrils consisting of otherwise globular, roughly natively folded protein. An activated complement system and cellular activation perpetuate these reactions which due to the affinity of ß(2)m-amyloid for the collagen of synovial surfaces result in the DRA syndrome.