Plasma proteome profiling identifies changes associated to AD but not to FTD.

BACKGROUND: Frontotemporal dementia (FTD) is caused by frontotemporal lobar degeneration (FTLD), characterized mainly by inclusions of Tau (FTLD-Tau) or TAR DNA binding43 (FTLD-TDP) proteins. Plasma biomarkers are strongly needed for specific diagnosis and potential treatment monitoring of FTD. We aimed to identify specific FTD plasma biomarker profiles discriminating FTD from AD and controls, and between FTD pathological subtypes. In addition, we compared plasma results with results in post-mortem frontal cortex of FTD cases to understand the underlying process. METHODS: Plasma proteins (n = 1303) from pathologically and/or genetically confirmed FTD patients (n = 56; FTLD-Tau n = 16; age = 58.2 ± 6.2; 44% female, FTLD-TDP n = 40; age = 59.8 ± 7.9; 45% female), AD patients (n = 57; age = 65.5 ± 8.0; 39% female), and non-demented controls (n = 148; 61.3 ± 7.9; 41% female) were measured using an aptamer-based proteomic technology (SomaScan). In addition, exploratory analysis in post-mortem frontal brain cortex of FTD (n = 10; FTLD-Tau n = 5; age = 56.2 ± 6.9, 60% female, and FTLD-TDP n = 5; age = 64.0 ± 7.7, 60% female) and non-demented controls (n = 4; age = 61.3 ± 8.1; 75% female) were also performed. Differentially regulated plasma and tissue proteins were identified by global testing adjusting for demographic variables and multiple testing. Logistic lasso regression was used to identify plasma protein panels discriminating FTD from non-demented controls and AD, or FTLD-Tau from FTLD-TDP. Performance of the discriminatory plasma protein panels was based on predictions obtained from bootstrapping with 1000 resampled analysis. RESULTS: Overall plasma protein expression profiles differed between FTD, AD and controls (6 proteins; p = 0.005), but none of the plasma proteins was specifically associated to FTD. The overall tissue protein expression profile differed between FTD and controls (7-proteins; p = 0.003). There was no difference in overall plasma or tissue expression profile between FTD subtypes. Regression analysis revealed a panel of 12-plasma proteins discriminating FTD from AD with high accuracy (AUC: 0.99). No plasma protein panels discriminating FTD from controls or FTD pathological subtypes were identified. CONCLUSIONS: We identified a promising plasma protein panel as a minimally-invasive tool to aid in the differential diagnosis of FTD from AD, which was primarily associated to AD pathophysiology. The lack of plasma profiles specifically associated to FTD or its pathological subtypes might be explained by FTD heterogeneity, calling for FTD studies using large and well-characterize cohorts.

Brain endothelial cell expression of SPARCL-1 is specific to chronic multiple sclerosis lesions and is regulated by inflammatory mediators in vitro.

AIMS: Cell matrix modulating protein SPARCL-1 is highly expressed by astrocytes during CNS development and following acute CNS damage. Applying NanoLC-MS/MS to CSF of RRMS and SPMS patients, we identified SPARCL-1 as differentially expressed between these two stages of MS, suggesting a potential as CSF biomarker to differentiate RRMS from SPMS and a role in MS pathogenesis. METHODS: This study examines the potential of SPARCL-1 as CSF biomarker discriminating RRMS from SPMS in three independent cohorts (n = 249), analyses its expression pattern in MS lesions (n = 26), and studies its regulation in cultured human brain microvasculature endothelial cells (BEC) after exposure to MS-relevant inflammatory mediators. RESULTS: SPARCL-1 expression in CSF was significantly higher in SPMS compared to RRMS in a Dutch cohort of 76 patients. This finding was not replicated in 2 additional cohorts of MS patients from Sweden (n = 81) and Switzerland (n = 92). In chronic MS lesions, but not active lesions or NAWM, a vessel expression pattern of SPARCL-1 was observed in addition to the expression by astrocytes. EC were found to express SPARCL-1 in chronic MS lesions, and SPARCL-1 expression was regulated by MS-relevant inflammatory mediators in cultured human BEC. CONCLUSIONS: Conflicting results of SPARCL-1's differential expression in CSF of three independent cohorts of RRMS and SPMS patients precludes its use as biomarker for disease progression. The expression of SPARCL-1 by BEC in chronic MS lesions together with its regulation by inflammatory mediators in vitro suggest a role for SPARCL-1 in MS neuropathology, possibly at the brain vascular level.

CSF neurofilament and N-acetylaspartate related brain changes in clinically isolated syndrome.

BACKGROUND: Axonal damage is considered a major cause of disability in multiple sclerosis (MS) and may start early in the disease. Specific biomarkers for this process are of great interest. OBJECTIVE: To study if cerebrospinal fluid (CSF) biomarkers for axonal damage reflect and predict disease progression already in the earliest stages of the disease, that is, in clinically isolated syndrome (CIS). METHODS: We assessed CSF levels of neurofilament heavy (NFH), neurofilament light (NFL) and N-acetylaspartate (NAA) in 67 patients with CIS and 18 controls with neuropsychiatric diseases of non-inflammatory aetiology (NC). Patients with CIS underwent baseline magnetic resonance imaging (MRI) at 3T, and a follow-up MRI after 1 year was obtained in 28 of them. RESULTS: Compared with NC, patients with CIS had higher NFH (p=0.05) and NFL (p<0.001) levels. No significant group differences were found for NAA. Patients' NFH levels correlated with physical disability (r=0.304, p<0.05) and with change in brain volume over 1 year of follow-up (r=-0.518, p<0.01) but not with change in T2 lesion load. CONCLUSION: Our results confirm increased neurofilament levels already in CIS being related to the level of physical disability. The association of NFH levels with brain volume but not lesion volume changes supports the association of these markers with axonal damage.

Identification of biomarkers for diagnosis and progression of MS by MALDI-TOF mass spectrometry.

INTRODUCTION: Body fluid biomarkers for clinical subtyping and monitoring of disease progression are of considerable interest in multiple sclerosis (MS). Proteomics tools are optimal for the unbiased simultaneous detection of large series of peptides and proteins. OBJECTIVES: To identify novel candidate biomarkers discriminating patients with MS from patients with other neurological diseases (OND), and for subtyping of relapsing-remitting (RR), secondary progressive (SP) and primary progressive (PP) MS patients using a high-throughput MALDI-TOF-based mass spectrometry method. METHODS: Paired cerebrospinal fluid (CSF) and serum samples of 41 RRMS, 30 SPMS, 13 PPMS patients and 25 patients with OND were analysed. RESULTS: Out of a total of 100 detected peptides in CSF and 200 peptides in serum, 11 peptides were differentially regulated in serum and two in CSF between patients with MS and the OND control group. Eleven peptides were differentially regulated in both serum and CSF between relapse-onset MS and PPMS patients. Lastly, four peptides were differentially regulated in serum and two in CSF between RRMS and SPMS patients. Specific peaks regulated in MS were tentatively identified as fragments of secretogranin III and complement C3. The peak intensity of the CSF peptide ion with m/z value 8607.7 correlated to atrophy (r = -0.27, p < 0.005), black hole volumes (r = 0.31, p < 0.008) and total lesion load (r = 0.34, p < 0.003). A serum peptide with m/z value of 872.4 elevated in SPMS correlated to Expanded Disability Status Scale (r = 0.341, p < 0.005) and atrophy (r = -0.286, p < 0.028). CONCLUSIONS: Using high-throughput body fluid profiling by MALDI-TOF mass spectrometry, small proteins and peptides were detected as promising candidate biomarkers for diagnosis and disease progression of MS.

Combination of CSF N-acetylaspartate and neurofilaments in multiple sclerosis.

OBJECTIVE: Axonal degeneration is the likely cause of disease progression in multiple sclerosis (MS). Our previous results indicated that neuron-specific N-acetylaspartate (NAA) is a candidate CSF biomarker for disease progression in MS. The aim of this study was to explore the potential of NAA as an early biomarker of axonal damage in MS. Next, we wanted to know the additional value of measurement of NAA compared to other candidate markers for axonal damage, such as neurofilament subunits and tau protein. METHODS: Levels of NAA, neurofilament light, neurofilament heavy, and tau were determined in CSF of patients with clinically isolated syndrome (CIS, n = 38), relapsing-remitting MS (RRMS, n = 42), secondary progressive MS (SPMS, n = 28), and primary progressive MS (PPMS, n = 6); patients without neurologic disease (ND, n = 28); noninflammatory neurologic controls (n = 18); and inflammatory neurologic controls (n = 39). RESULTS: CSF NAA levels were decreased in patients with SPMS compared to ND controls, patients with CIS, and patients with RRMS. CSF NAA levels in patients with CIS and RRMS were similar to those in ND subjects. All axonal damage proteins showed specific patterns of changes and relations with disease activity measures. The neurofilament light chain levels were already increased in patients with CIS, especially in patients who converted to MS. The neurofilament heavy chain levels were highest in the patients with SPMS. Tau levels were similar in MS and ND. CONCLUSIONS: CSF N-acetylaspartate (NAA) levels were not different from patients without neurologic disease in early stages of multiple sclerosis, though decreased as the disease progressed. Combining CSF NAA and neurofilament levels yields information on different phases of axonal pathology.

A study on associations between antiprothrombin antibodies, antiplasminogen antibodies and thrombosis.

Anti-prothrombin antibodies are a frequent cause of lupus anticoagulant (LAC), a thrombotic risk factor. Prothrombin shares structural homology with plasminogen, a kringle protein with an important role in fibrinolysis. Cross-reactivity between antiprothrombin antibodies and plasminogen has been described. To study associations between LAC, IgG and IgM class antiprothrombin and antiplasminogen antibodies, plasminogen activity levels and thrombosis in selected patients with systemic autoimmune diseases. Patients included forty-six consecutive LAC-positive patients (29 with systemic lupus erythematosus (SLE); 33 with a thrombotic history), 38 patients without LAC (36 with SLE; seven with a history of thrombosis) and 40 healthy controls. In the total group of 84 patient samples, the prevalence of antiprothrombin and antiplasminogen antibodies was 30 and 38%, respectively. There was no significant relationship between the presence of these antibodies. In contrast to presence of antiplasminogen antibodies, presence of antiprothrombin antibodies was statistically significant related to thrombosis. Thirteen samples had antiprothrombin and antiplasminogen antibodies of similar isotype (IgG, n= 4; IgM, n= 9). Of these, all but one had LAC and 11/13 came from patients with a history of thrombosis. Simultaneous presence of IgM-class antiprothrombin and antiplasminogen antibodies had a significant association with thrombosis. Levels of plasminogen activity were similar in samples from healthy controls and patients (with or without antiplasminogen antibodies or thrombosis). Anti-prothrombin antibodies and antiplasminogen antibodies occur frequently in patients with systemic autoimmune disease. Anti-prothrombin antibodies, but not antiplasminogen antibodies are a risk factor for thrombosis. Anti-plasminogen are in most cases unrelated to antiprothrombin antibodies.

A simple method to discriminate between beta2-glycoprotein I- and prothrombin-dependent lupus anticoagulants.

Lupus anticoagulants (LAC) are a heterogeneous group of autoantibodies that prolong phospholipid-dependent clotting assays. The autoantibodies that cause LAC activity are predominantly directed against beta2-glycoprotein I (beta2GPI) or prothrombin. In the present study, we describe a method to differentiate between LAC caused by antibodies directed against beta2GPI or prothrombin. Monoclonal antibodies, affinity purified patient antibodies, and selected patient samples were used to show that in an aPTT-based clotting assay (PTT-LA; Diagnostica Stago), the use of cardiolipin vesicles in the neutralization procedure discriminates between beta2GPI- or prothrombin-dependent LAC activities. Addition of cardiolipin vesicles shortened the prolonged clotting time caused by anti-beta2GPI antibodies with LAC activity, whereas this procedure further prolonged clotting times caused by antiprothrombin antibodies with LAC activity. In contrast, addition of phosphatidylcholine/phosphatidylserine vesicles corrected prolonged clotting times caused by either anti-beta2GPI or antiprothrombin antibodies with LAC activity. The effects of cardiolipin (CL) on beta2GPI-induced LAC activity were specific for contact activation mediated clotting assays. Possible explanations for these findings are the relatively high affinity of beta2GPI for cardiolipin, as determined by surface plasmon resonance analysis, and inhibition by anti-beta2GPI antibodies of the CL-induced prolongation of the PTT-LA.

Complexes of anti-prothrombin antibodies and prothrombin cause lupus anticoagulant activity by competing with the binding of clotting factors for catalytic phospholipid surfaces.

We investigated the mechanism by which anti-prothrombin antibodies cause lupus anticoagulant (LAC) activity. Addition of affinity-purified anti-prothrombin antibodies from LAC-positive plasma samples (alpha-FII-LAC+) to normal plasma induced LAC activity. Upon increasing the phospholipid concentration, LAC activity was neutralized. Addition of purified alpha-FII-LAC+ to normal plasma strongly inhibited factor Xa formation. No inhibition was measured when alpha-FII-LAC+ were added to prothrombin-deficient plasma or when purified anti-prothrombin antibodies from LAC-negative plasma samples (alpha-FII-LAC-) were added. When a combination of prothrombin and alpha-FII-LAC+ was added to the purified clotting complex, a strong inhibition of factor Xa and IIa formation was seen. The alpha-FII-LAC+ alone or a combination of prothrombin and alpha-FII-LAC- did not show inhibition. Ellipsometry studies showed that, in the presence of alpha-FII-LAC+, the affinity of prothrombin for a phospholipid surface increased dramatically, whereas a much lower increase was observed with alpha-FII-LAC-. Our results show that complexes of prothrombin and anti-prothrombin antibodies with LAC activity inhibit both prothrombinase and tenase. The antibodies increase the affinity of prothrombin for the phospholipid surface, thereby competing with clotting factors for the available catalytic phospholipid surface, a mechanism similar to that of anti-beta2-glycoprotein I antibodies.

Structure-function studies on beta 2-glycoprotein I.

Human beta 2-glycoprotein I is a heavily glycosylated plasma protein which has been implicated in the binding of antiphospholipid antibodies to negatively charged phospholipids; a process considered as an important risk factor for the development of thrombosis. We have solved the crystal structure of beta 2-glycoprotein I. In this review we will discuss what the three-dimensional structure teaches us about the role of beta 2-glycoprotein I in the pathogenesis of the antiphospholipid syndrome.

Induction of functional IL-8 receptors by IL-4 and IL-13 in human monocytes.

IL-8 and related Glu-Leu-Arg (ELR+) CXC chemokines are potent chemoattractants for neutrophils but not for monocytes. IL-13 and IL-4 strongly increased CXCR1 and CXCR2 chemokine receptor expression in human monocytes, macrophages, and dendritic cells. The effect was receptor- and cell type-selective, in that CCRs were not increased and no augmentation was seen in neutrophils. The effect was rapid, starting at 4 h, and concentration dependent (EC50 = 6.2 and 8.3 ng/ml for CXCR1 and CXCR2, respectively) and caused by new transcriptional activity. IL-13/IL-4-treated monocytes showed increased CXCR1 and CXCR2 membrane expression. IL-8 and related ELR+ chemokines were potent and effective chemotactic agents for IL-13/IL-4-treated monocytes, but not for untreated mononuclear phagocytes, with activity comparable to that of reference monocyte attractants, such as MCP-1. In the same cells, IL-8 also caused superoxide release. Macrophages and dendritic cells present in biopsies from Omenn's syndrome and atopic dermatitis patients, two Th2 skewed pathologies, expressed IL-8 receptors by immunohistochemistry. These results show that IL-13 and IL-4 convert IL-8 and related ELR+ chemokines, prototypic neutrophil attractants, into monocyte chemotactic agonists, by up-regulating receptor expression. Therefore, IL-8 and related chemokines may contribute to the accumulation and positioning of mononuclear phagocytes in Th2-dominated responses.

Adhesion mechanism of human beta(2)-glycoprotein I to phospholipids based on its crystal structure.

Human beta(2)-glycoprotein I is a heavily glycosylated five-domain plasma membrane-adhesion protein, which has been implicated in blood coagulation and clearance of apoptotic bodies from the circulation. It is also the key antigen in the autoimmune disease anti-phospholipid syndrome. The crystal structure of beta(2)-glycoprotein I isolated from human plasma reveals an elongated fish-hook-like arrangement of the globular short consensus repeat domains. Half of the C-terminal fifth domain deviates strongly from the standard fold, as observed in domains one to four. This aberrant half forms a specific phospholipid-binding site. A large patch of 14 positively charged residues provides electrostatic interactions with anionic phospholipid headgroups and an exposed membrane-insertion loop yields specificity for lipid layers. The observed spatial arrangement of the five domains suggests a functional partitioning of protein adhesion and membrane adhesion over the N- and C-terminal domains, respectively, separated by glycosylated bridging domains. Coordinates are in the Protein Data Bank (accession No. 1QUB).

Anti-prothrombin antibodies and their relation with thrombosis and lupus anticoagulant.

Antiphospholipid antibodies are a heterogeneous group of antibodies, comprising antibodies with different antigen specificity. Prothrombin is one of the antigens which can be detected by antiphospholipid antibodies and therefore anti-prothrombin antibodies belong to the antiphospholipid antibody family. The presence of antiphospholipid antibodies correlates strongly with thromboembolic complications; however a mechanism by which these autoantibodies induce a thrombotic complication in vivo is not understood. The classic assays for the detection of antiphospholipid antibodies (LAC and anticardiolipin ELISAs) aim to measure all the antiphospholipid antibodies present in the samples without making a distinction between the different subspecificities of the antibodies present in one single sample. Moreover, most of the in-vitro studies performed were carried out with total IgGs, which contain a mixture of antibodies. The absence of an accurate characterization of the plasma samples and the lack of specificity of the IgGs used in in-vitro tests makes it difficult to determine the contribution of antiprothrombin antibodies to the thrombotic complications. Here we review and critically analyse the literature regarding the clinical relevance of the presence of antiprothrombin antibodies and the possible participation of these antibodies in the pathogenesis of the thrombotic complications.

Effect of intraperitoneal administration of granulocyte/macrophage-colony-stimulating factor in rats on omental milky-spot composition and tumoricidal activity in vivo and in vitro.

Milky spots in the greater omentum are small accumulations of leucocytes that consist mainly of macrophages and have recently shown to be a selective dissemination site of intraperitoneal (i.p.) inoculated tumour cells. However, milky-spot macrophages show tumoricidal activity and may, therefore, be an excellent source of effector cells suited for local immunotherapy. In the present study we first examined whether granulocyte/macrophage-colony-stimulating factor (GM-CSF) treatment of isolated milky-spot macrophages affects the cytotoxicity against syngeneic colon carcinoma cells (CC531) in vitro. Secondly, we studied the influence of intraperitoneal GM-CSF administration on the number and antitumour activity of milky-spot and peritoneal macrophages. All studies were performed in Wag/Rij rats in which a syngeneic colon carcinoma cell line (CC531) is available. The results of the in vitro study showed that GM-CSF treatment of the omental macrophages led to an increased cytotoxicity against the tumour cell line. Intraperitoneal administration of 1000 U GM-CSF daily for 7 consecutive days demonstrated both an enhanced antitumour activity of the milky-spot macrophages and an increase in the milky-spot macrophage population. An increase in the proliferative capacity, according to bromodeoxyuridine incorporation, was shown in the milky-spot macrophages. Taking into account both the enhanced macrophage number and their enhanced activity upon i.p. GM-CSF treatment, the milky-spot macrophages may provide a rationale for local intraperitoneal immunotherapy in the prevention of intra-abdominal tumour growth.