Microparticles expressing myeloperoxidase as potential biomarkers in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV).

To investigate presence of circulating myeloperoxidase-positive microparticles (MPO+MPs) in relation to disease activity in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Forty-six patients with AAV and 23 age- and sex-matched healthy controls were included. Vasculitis disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS). MPs were analyzed in citrate plasma by flow cytometry and phenotyped based on MPO expression and co-expression of pentraxin-3 (PTX3), high mobility group box 1 protein (HMGB1), and tumor necrosis factor-like weak inducer of apoptosis (TWEAK). Serum levels of PTX3, sTWEAK, and HMGB1 were also determined. Twenty-three patients had active vasculitis (BVAS ≥ 1). Concentrations of MPO+MPs expressing PTX3, HMGB1, and TWEAK were significantly higher in patients compared to healthy controls (p < 0.001, p < 0.01, p < 0.001, respectively), while concentrations of PTX3+ and HMGB1+MPO+MPs were significantly higher in active AAV compared to patients in remission. MPO+MPs expressing either PTX3 or HMGB1 were associated with BVAS (r = 0.5, p < 0.001; r = 0.3, p = 0.04, respectively). Significantly higher serum PTX3 levels were found in active- than in inactive AAV (p < 0.001), correlating strongly with BVAS (r = 0.7, p < 0.001). Serum levels of sTWEAK and HMGB1 did not differ between patients and controls. Concentration of MPO+MPs is increased in plasma from AAV patients compared to healthy individuals. PTX3 in serum as well as PTX3 and HMGB1 expressed on MPO+MPs were associated with disease activity in the investigated patients. KEY MESSAGES: Myeloperoxidase-positive microparticles (MPO+MPs) are increased in plasma from patients with ANCA-associated vasculitis. Concentrations of MPO+MPs expressing PTX3, HMGB1, and TWEAK were significantly higher in patients compared to healthy controls. MPO+MPs expressing PTX3 and HMGB1 are associated with disease activity in ANCA-associated vasculitis.

Brain-Derived Microparticles in Patients with Severe Isolated TBI.

PRIMARY OBJECTIVE: to investigate the presence of circulating microparticles (MPs) of brain tissue origin in the systemic and cerebrovenous blood of patients with severe traumatic brain injury (TBI). RESEARCH DESIGN: Prospective observational study in 15 consecutive patients with severe isolated TBI. METHODS AND PROCEDURES: We repeatedly measured concentrations of MPs expressing glial fibrillary acidic protein (GFAP), neuron-specific enolase (NSE) and aquaporin-4 (AQP4), in arterial and cerebrovenous blood at admittance to hospital and up to 72 hours after the injury. MAIN OUTCOMES AND RESULTS: Concentrations of MPs expressing GFAP and AQP4 were significantly higher in the TBI group compared with healthy controls: GFAP 2.0 [1.1-7.9] vs. 1.3 [1-2.1] × 106/mL, p < 0.001; AQP4 0.1 [0.07-0.22] vs. 0.08 [0.06-0.11] × 106/mL, p < 0.001 (median, range). No transcranial gradients were found. Levels of NSE-expressing MPs were also higher in the TBI group compared with healthy controls: 0.4 [0.25-2.1] vs. 0.26 [0.13-0.98] × 106/mL, p < 0.05; however, regarding NSE-positive non-platelet MPs, there were no differences between patients and controls. CONCLUSIONS: Patients with TBI have higher numbers of brain-derived MPs. Further studies are needed, however, to identify specific and sensitive MP markers of brain injury.

Altered ß2 -glycoprotein I expression on microparticles in the presence of antiphospholipid antibodies.

Essentials ß2 glycoprotein-I (ß2 GPI) is a scavenger molecule that binds to microparticles (MPs). ß2 GPI expression on MPs was measured in systemic lupus erythematosus (SLE) patients and controls. ß2 GPI positive MPs is depressed among SLE patients positive for antiphospholipid antibodies. Complex formation between ß2 GPI on MPs and patients own anti-ß2 GPI may disturb MP clearance. Click to hear an ISTH Academy presentation on antiphospholipid antibody syndrome by Drs de Laat and Bertolaccini SUMMARY: Background Antiphospholipid antibodies (aPLs) together with thrombosis and/or pregnancy morbidities characterize the antiphospholipid syndrome. ß2 -Glycoprotein I (ß2 GPI), the most important antigen for aPLs, is a scavenger molecule that specifically binds to phosphatidylserine (PS) expressed on microparticles (MPs). Objectives To evaluate ß2 GPI-expressing MPs in patients with systemic lupus erythematosus (SLE) stratified for aPL status, and in healthy controls. Patients/Methods We investigated 18 aPL/anti-ß2 GPI-positive and 22 aPL-negative patients from a large SLE cohort and 19 healthy controls. ß2 GPI-positive MPs and IgG-positive MPs were detected by flow cytometry. We measured plasma levels of ß2 GPI, and performed in vitro experiments to investigate the binding properties of ß2 GPI on MPs. Results SLE patients had more MPs and IgG-positive MPs than controls. We observed fewer ß2 GPI-positive MPs in aPL/anti-ß2 GPI-positive patients than in aPL/anti-ß2 GPI-negative patients and controls (approximately two-fold). ß2 GPI levels in plasma did not differ with aPL/anti-ß2 GPI status in patients; however, controls had slightly higher levels of ß2 GPI than aPL/anti-ß2 GPI-positive patients. In vitro experiments revealed that ß2 GPI preferentially binds to PS-positive MPs. Conclusions Despite abundant total MPs and MPs in immune complexes, ß2 GPI-positive MPs were depleted in SLE patients, and the levels were especially low in aPL/anti-ß2 GPI-positive patients. We suggest that anti-ß2 GPI antibodies bind to ß2 GPI-PS complexes expressed on MPs. Consequent loss of ß2 GPI-PS expression on MPs may impair scavenging and contribute to the accumulation of circulating PS-negative MPs, a possible source of autoantigens. Autoantibodies delaying MP clearance may thus constitute an important mechanism underlying autoimmunity.

High interleukin-6 and impulsivity: determining the role of endophenotypes in attempted suicide.

The dysregulation of inflammation has been associated with depression and, more recently, with suicidal behaviors. The reports regarding the relationship between interleukin-6 (IL-6) and suicide attempts are inconsistent. Personality traits such as impulsivity and aggression are considered endophenotypes and important factors that underlie suicidal behaviors. The aim of the current study was to assess whether plasma and cerebrospinal fluid (CSF) levels of IL-6 are associated with personality traits among suicide attempters. We assessed the relationships among personality traits, IL-6 and violent suicide attempts. The plasma and CSF levels of IL-6 were measured in suicide attempters (plasma=58, CSF=39) using antibody-based immunoassay systems. Personality domains were assessed using the Karolinska Scale of Personality (KSP). IL-6 levels in plasma and CSF were used to predict personality domains via regression models. Plasma IL-6 was significantly and positively correlated with extraversion as well as the KSP subscales impulsivity and monotony avoidance. CSF IL-6 was positively correlated with monotony avoidance. Violent suicide attempts tended to be associated with high plasma IL-6 levels. Plasma and CSF levels of IL-6 were not significantly associated with each other. These results indicate that impulsivity and the choice of a violent suicide attempt method might be related to higher levels of IL-6 in individuals who attempt suicide. The neuroinflammation hypothesis of suicidal behavior on the basis of elevated IL-6 levels might be partly explained by the positive association between IL-6 and impulsivity, which is a key element of the suicidal phenotype.

The expression of HMGB1 on microparticles from Jurkat and HL-60 cells undergoing apoptosis in vitro.

HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane-bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL-60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze-thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.

Effect of lipopolysaccharide administration on the number, phenotype and content of nuclear molecules in blood microparticles of normal human subjects.

Microparticles (MPs) are small membrane-bound vesicles that arise from activated and dying cells and promote inflammation and thrombosis. To characterize the in vivo release of MPs, we used flow cytometry to measure MPs in the blood of 15 healthy volunteers administered bacterial endotoxin (lipopolysaccharide or LPS) in the presence of a low dose of hydrocortisone with or without inhaled nitric oxide. MPs, defined as particles less than 1.0 µm in size, were assessed following labelling for CD42a, CD14 and CD62E or CD144 antibodies to identify MPs from platelets (PMPs), monocytes (MMPs) and endothelial cells (EMPs). In addition, PMPs and MMPs were labelled with anti-HMGB1 and stained with SYTO13 to assess nuclear acid content. Administration of LPS led to an increase in the numbers of PMPs, MMPs and EMPs as defined by CD62E, as well as the number of MMPs and PMPs staining with anti-HMGB1 and SYTO13. Inhalation of NO did not influence these findings. Together, these studies show that LPS can increase levels of blood MPs and influence phenotype, including nuclear content. As such, particles may be a source of HMGB1 and other nuclear molecules in the blood during inflammation.

Is a decrease of microparticles related to improvement of hemostasis after FVIII injection in hemophilia A patients treated on demand?

BACKGROUND: Microparticles (MPs) are small membrane vesicles (0.1-1 µm) released from various cells after activation and/or apoptosis. There are limited data about their role in hemophilia A. PATIENTS AND METHODS: Blood samples were taken before and 30 min after FVIII injection in 18 patients with severe hemophilia A treated on demand. Flow-cytometric determination of total MPs (TMPs) using lactadherin, platelet MPs (PMPs) (CD42a), endothelial MPs (EMPs) (CD144) and leukocyte MPs (LMPs) (CD45) was performed. The results were compared with data on endogenous thrombin potential (ETP), overall hemostatic potential (OHP), fibrin gel permeability and thrombin-activatable fibrinolysis inhibitor (TAFI). RESULTS AND CONCLUSIONS: TMPs and PMPs decreased after treatment (to 1015 ± 221 [SEM] and 602 ± 134 × 10(6)  L(-1) ) in comparison with values before treatment (2373 ± 618 and 1316 ± 331; P < 0.01). EMPs also decreased after treatment (78 ± 12 vs. 107 ± 13; P < 0.05) while LMPs were not influenced. Both TMP and PMP counts were inversely correlated, moderately but statistically significantly, with data on OHP, ETP, fibrin network permeability and TAFI/TAFIi (P < 0.05 for all). EMP counts were correlated only with ETP (P < 0.05), while LMP counts did not show any correlation. TMP and PMP counts were also inversely correlated with FVIII levels (P < 0.05). TMP, PMP and EMP counts decreased after on-demand treatment with FVIII concentrate in hemophilia A patients. The decrease in circulating MPs, which were inversely correlated with hemostatic activation, may imply that MPs are incorporated in the hemostatic plug formed after FVIII substitution at the site of injury.

Low vascular endothelial growth factor and interleukin-8 in cerebrospinal fluid of suicide attempters.

A dysregulated immune system influencing pathways for cytokine regulation and growth factor expression is implicated in the pathophysiology of several neuropsychiatric disorders. Here, we analyzed cerebrospinal fluid (CSF) cytokines and growth factors with an ultra-sensitive immunoassay system in 43 medication-free suicide attempters and 20 healthy male volunteers. CSF vascular endothelial growth factor (VEGF) and CSF interleukin-8 (IL-8) levels were significantly lower in suicide attempters compared with healthy controls. Further, CSF VEGF showed a significant negative correlation with depression severity. CSF IL-6 levels did not differ between suicide attempters and healthy controls. Low CSF levels of VEGF may represent a lack of trophic support to neurons and downregulation of neurogenesis in the hippocampus reflecting more severe depressive states. IL-8 has also been reported as important in neuroprotection as well as having chemokine activity in the innate immune response. The results support a role for an impaired innate immunity and dysregulation of neuroprotection in the pathophysiology of depression and suicidal behavior.

Studies of microparticles in patients with the antiphospholipid syndrome (APS).

OBJECTIVES: To study circulating platelet, monocyte and endothelial microparticles (PMPs, MMPs and EMPs) in patients with antiphospholipid syndrome (APS) in comparison with healthy controls. MATERIAL AND METHOD: Fifty-two patients with APS and 52 healthy controls were investigated. MPs were measured on a flow cytometer (Beckman Gallios) and defined as particles sized < 1.0 µm, negative to phalloidin, positive to lactadherin and positive to either CD42a (PMPs), CD144 (EMPs) or CD14 (MMPs). Exposure of CD142 (TF) was measured on CD144 positive MPs. RESULTS: Total number of MPs (i.e. lactadherin positive particles) was higher in APS patients versus controls (p < 0.001). An increased number of EMPs (p < 0.001), increased TF-positive EMPs (p < 0.001) and increased MMPs (p < 0.001) were also observed. PMP numbers did not differ between the groups. None of the MP types differed in numbers between obstetric and thrombotic APS patients. CONCLUSION: We observed a high number of EMPs expressing TF in APS patients. The numbers of MMPs and total EMPs were also higher as compared with healthy controls but in contrast to previous reports, the number of PMPs did not differ between groups.

On-demand but not prophylactic treatment with FVIII concentrate increase thrombin activatable fibrinolysis inhibitor activation in severe haemophilia A patients.

INTRODUCTION: Thrombin activatable fibrinolysis inhibitor (TAFI) down-regulates fibrinolysis after activation by thrombin/thrombomodulin. We investigated the effect of treatment with FVIII concentrate on plasma levels of pro-TAFI and activated TAFI in haemophilia A patients. METHODS: Samples were collected pre and posttreatment from patients treated prophylactically or on-demand. Pro-TAFI, TAFI/TAFIi and FVIII levels were measured in all samples. RESULTS: Treatment had no effect on pro-TAFI levels. Pro-TAFI was similar in both patient groups but higher than in controls. Patients from the prophylactic treatment group had measurable FVIII levels pretreatment while in the treatment-on-demand group FVIII levels were ≤0.01 IU/mL. In the prophylactic treatment group, the levels of TAFI/TAFIi were significantly lower pre- and posttreatment (4.31 ± 3.14 and 3.48 ± 2.65 ng/mL respectively) than in the on-demand group (13.02 ± 3.47 and 14.87 ± 3.47 ng/mL respectively). This difference may be due to release of tissue factor at the injury site in the on-demand group. This could induce thrombin and TAFI activation within the clot counterbalancing fibrinolysis in these patients. In the prophylactic group, no injury existed, thus there was insufficient thrombin generation within the clot to activate TAFI. CONCLUSION: These findings suggest that in patients to whom FVIII is administered on demand the fibrinolysis activity is more down regulated than in patients following a prophylactic treatment regime.