Relationship between Inflammation and Aspirin and Clopidogrel Antiplatelet Responses in Acute Ischemic Stroke.

OBJECTIVE: We measured serum levels of proinflammatory/prothrombotic markers P-selectin, CD40L, matrix metalloproteinase 9 (MMP-9), intracellular adhesion molecule 1 (ICAM-1), and interleukin (IL)-6 in ischemic stroke patients, correlating their levels with the results of aspirin (ASA) and clopidogrel antiplatelet responses, using 3 "point of care" platelet function instruments, thromboelastograph (TEG), Accumetrics (ACU), and impedance aggregometer (IMP). METHODS: Patients on chronic ASA regimen at the time of stroke were switched to 300 mg clopidogrel loading dose and 75 mg clopidogrel maintenance dose. Serum levels of the aforementioned inflammatory mediators were measured in 51 patients at baseline (on ASA regimen), and at 26 ± 5 hours and 64 ± 18 hours postclopidogrel administration by enzyme-linked immunosorbent assay. RESULTS: P-selectin, CD40L, and MMP-9 serum levels were reduced; ICAM-1 and IL-6 serum levels showed no difference postclopidogrel administration relative to baseline. Patients' stratification based on ASA dose showed more significant reductions in P-selectin, CD40L, and MMP-9 serum levels postclopidogrel administration in patients who were on baseline 81 mg ASA, as compared to patients on 325 mg ASA. Measurement with TEG was sensitive for correlating ASA antiplatelet responses to serum levels of inflammatory markers, whereas measurements with ACU and IMP were sensitive for correlating clopidogrel antiplatelet responses to serum levels of inflammatory markers. CONCLUSION: Clopidogrel exerts both platelet-dependent and platelet-independent anti-inflammatory effects. The association between platelet function and inflammation depends on the platelet function analyzer, the type of antiplatelet agent, the nature of the inflammatory marker, and the time of measurement relative to the time of drug administration.

Quantitative and qualitative pleiotropic differences between Simvastatin single and Vytorin combination therapy in hypercholesterolemic subjects.

AIMS: This cross-sectional study tested the hypothesis that treatment with the combination of Ezetimibe/Simvastatin (Vytorin) leads to broader changes in the expression levels of immunomodulatory genes as compared to Simvastatin monotherapy. METHODS: Illumina's GenomeStudio gene expression module was used to compare gene profiles of Vytorin and Simvastatin in the peripheral blood mononuclear cells of 20 hypercholesterolemic subjects. RESULTS: The characteristics of the immunomodulatory genes, which were altered by Vytorin, differed from those genes which were altered by Simvastatin. Vytorin mostly altered the expression levels of genes related to inflammation/oxidative stress; it downregulated the NF-KappaB and upregulated the expression of anti-inflammatory cytokine, IL-10, and anti-oxidant enzymes, GPX1 and SOD2, but also upregulated the expression levels of genes involved in cellular activation, adhesion, and coagulation cascade, including VWF, F7, PF4, PF4V1 SELP, ITGB3, ITGB5. Simvastatin mostly altered the expression levels of genes related to cellular apoptosis/proliferation. It upregulated the expression levels of apoptosis-related genes APAF1, BAX, IER3, and CSF1R, and downregulated the expression levels of genes related to cellular proliferation, including PTN and CD69. Treatment with Vytorin combination therapy modulated lipid profile and serum levels of the C-reactive protein more effectively, than treatment with Simvastatin monotherapy. CONCLUSION: The nature of the pleiotropic effects may play a role in Vytorin's and Simvastatin's clinical efficacies.

Flow cytometry and gene expression profiling of immune cells of the carotid plaque and peripheral blood.

OBJECTIVES: The relative contribution of the local vs. peripheral inflammation to the atherothrombotic processes is unknown. We compared the inflammatory status of the immune cells of the carotid plaque with similar cells in peripheral circulation of patients with advanced carotid disease (PCDs). METHODS: Mononuclear cells (MNCs) were extracted from carotid endarterectomy (CEA) samples by enzymatic digestion and subsequent magnetic cell sorting. The cell surface antigenic expressions, and mRNA expression levels were compared between CEA MNCs and peripheral MNCs, using flow cytometry and RT-PCR techniques. RESULTS: The percentages of resting MNCs were lower, and activated MNCs, particularly monocytes, were higher in the CEAMNCs, as compared to the peripheral MNCs. The percentages of activated T cells and B cells were higher in the peripheral MNCs of PCDs, than in healthy controls (HCs), but the percentages of activated monocytes did not differ between the two groups. The expression levels of both pro-inflammatory/pro-thrombotic (P(38), JNKB-1, Egr-1 PAI-1, MCP-1, TF, MMP-9, HMGB-1, TNF-α, mTOR) and anti-inflammatory (PPAR-γ, TGF-ß) mediators were significantly higher in the CEA MNCs as compared to the peripheral MNCs. Furthermore, MMP-9 and PPAR-γ expression levels were higher in the peripheral MNCs of PCDs than HCs. CONCLUSION: The inflammatory status is higher in the immune cells of the carotid plaque, as compared to those cells in the peripheral blood. The altered expression levels of both pro-inflammatory/pro-thrombotic and anti-inflammatory mediators in the milieu of the plaque suggest that the balance between these various mediators may play a key role in carotid disease progression.

MRI characteristics of patients with antiphospholipid syndrome and multiple sclerosis.

MRI findings of primary anti-phospholipid antibody syndrome (PAPLS) are difficult to distinguish from those of multiple sclerosis (MS). Only a few previous studies have compared conventional and non-conventional MRI findings in MS and PAPLS patients. In addition, MRI differences between anti-phospholipid antibody (APLA) positive (+) and APLA negative (-) MS patients have not been reported. Therefore, the aim of this study was to investigate the differences in MRI measures among patients with PAPLS, MS and normal control (NC) subjects. We also explored non-conventional MRI measures in APLA+ and APLA- MS patients. Forty-nine (49) consecutive MS patients among whom 39 had relapsing-remitting (RR) and 10 secondary-progressive (SP) disease course, 30 patients with PAPLS and 49 NC were enrolled. Twenty-eight (28) MS patients were APLA+. MRI measures of T1- and T2-lesion volumes (LV) and brain atrophy, including fractions of whole brain (BPF), gray matter (GMF) and white matter (WMF), were evaluated. The magnetization transfer ratio (MTR) of T2- and T1-LVs and different normal-appearing brain tissue (NABT) compartments as well as diffusion-weighted imaging of whole brain mean parenchyma diffusivity (MPD) were obtained. MS patients differed significantly from NC in all MRI measures. PAPLS patients differed from NC in their T2-LV, in MTR measures and in MPD. When MS patients were compared to PAPLS patients, they showed significantly higher T2- and T1-LVs and T2-LV MTR, lower BPF and GMF and higher MPD. APLA+ RR and SPMS (all APLA+) patients showed significantly higher T2-LV, lower GMF, lower normal-appearing gray matter MTR and higher MPD when compared to APLA- patients. The results indicate that brain abnormalities can be detected in PAPLS patients with non-conventional MRI. MRI reveals more profound injury in patients with MS versus PAPLS. APLA mediates heterogeneous cerebral pathology that remains to be further investigated.

Immunomodulatory responses of peripheral blood mononuclear cells from multiple sclerosis patients upon in vitro incubation with the flavonoid luteolin: additive effects of IFN-beta.

The study is aimed to determine the role of luteolin (3',4',5,7-tetrahydroxyflavone), alone and in combination with human interferon-beta (IFN-beta), in modulating the immune response(s) of peripheral blood mononuclear cells (PBMCs) isolated from multiple sclerosis (MS) patients. PBMC proliferation in the presence or absence of these drugs was determined and the production of pro-inflammatory cytokines (IL-1beta, TNF-alpha), and the ratio of cell migration mediator MMP-9, and its inhibitor, TIMP-1 was assessed in the culture supernatants. Luteolin reduced, in a dose-dependent manner, the proliferation of PBMCs, and modulated the levels of IL-1beta and TNF-alpha released by PBMCs in the culture supernatants. Luteolin reduced the MMP-9/TIMP-1 ratio via lowering MMP-9 production. In the majority of cases, luteolin, when combined with IFN-beta, had additive effects in modulating cell proliferation, IL-1beta, TNF-alpha, MMP-9 and TIMP-1.

Diffusion-weighted imaging predicts cognitive impairment in multiple sclerosis.

Following a previous study with diffusion tensor imaging, we investigated the correlation between diffusion-weighted imaging (DWI) and cognitive dysfunction in multiple sclerosis (MS). We studied 60 MS patients (mean age 45.8+/-9.0 years) using 1.5-T MRI. Disease course was RR=40 and SP = 20. Mean disease duration was 12.8+/-8.7 years. Mean EDSS was 3.4+/-1.7. Whole brain, gray and white matter normalized volumes were calculated on 3D SPGR T1-WI using a fully automated Hybrid SIENAX method. Parenchymal mean diffusivity (PMD) maps were created after automated segmentation of the brain parenchyma and cerebrospinal fluid using T2-WI and DW images. Histogram analysis was performed and DWI indices of peak position (PP), peak height (PH), mean parenchymal diffusivity (MPD) and entropy were obtained. Neuropsychological (NP) evaluation emphasized auditory/verbal and visual/spatial memory, as well as processing speed and executive function. We found significant correlations between DWI and performance in all cognitive domains. Overall, stronger correlations emerged for MPD and entropy than other DWI measures, although all correlations were in the expected direction. The strongest association was between DWI entropy and performance on the Symbol Digit Modalities Test, which assesses processing speed and working memory (r = -0.54). Fisher r to z transformations revealed that DWI, gray matter (GMF) and whole brain (BPF) atrophy, T1-lesion volume (LV) and T2-LV all accounted for similar amounts of variance in NP testing. Stepwise regression models determined whether multiple MRI measures predicted unique additive variance in test performance. GMF (R2 = 0.35, F =30.82, P <0.01) and entropy (DeltaR2 =0.06, DeltaF=5.47, P <0.05) both accounted for unique variance in processing speed. Our data make a stronger case for the clinical validity of DWI in MS than heretofore reported. DWI has very short acquisition times, and the segmentation method applied in the present study is reliable and fully automated. Given its overall simplicity and moderate correlation with cognition, DWI may offer several logistic advantages over more traditional MRI measures when predicting the presence of NP impairment.

Quantitative diffusion weighted imaging measures in patients with multiple sclerosis.

Diffusion-weighted imaging (DWI) has been proposed as a sensitive measure of disease severity capable of detecting subtle changes in gray matter and white matter brain compartments in patients with multiple sclerosis (MS). However, DWI has been applied to the study of MS clinical subtypes in only a few studies. The objective of this study was to demonstrate the validity of a novel, fully automated method for the calculation of quantitative DWI measures. We also wanted to assess the correlation between whole brain (WB)-DWI variables and clinical and MRI measures of disease severity in a large cohort of MS patients. For this purpose we studied 432 consecutive MS patients (mean age 44.4+/-10.2 years), 16 patients with clinically isolated syndrome (CIS) and 38 normal controls (NC) using 1.5 T brain MRI. Clinical disease subtypes were as follows: 294 relapsing-remitting (RR), 123 secondary-progressive (SP) and 15 primary-progressive (PP). Mean disease duration was 12+/-10 years. Mean Expanded Disability Status Scale (EDSS) was 3.3+/-2.1. Brain parenchymal fraction (BPF), gray matter fraction (GMF) and white matter fraction (WMF) were calculated using a fully automated method. Mean parenchymal diffusivity (MPD) maps were created. DWI indices of peak position (PP), peak height (PH), MPD and entropy (ENT) were obtained. T2- and T1-lesion volumes (LV), EDSS, ambulation index (AI) and nine-hole peg test (9-HPT) were also assessed. MS patients had significantly lower BPF (d=1.26; p<0.001) and GMF (d=0.61; p=0.003), and higher ENT (d=1.2; p<0.0001), MPD (d=1.04; p<0.0001) and PH (d=0.47; p=0.045) than NC subjects. A GLM analysis, adjusted for age and multiple comparisons, revealed significant differences between different clinical subtypes for BPF, GMF, ENT, PH, PP, T2-LV and T1-LV (p<0.0001), WMF (p=0.001) and MPD (p=0.023). In RR and SP MS patients, ENT showed a more robust correlation with other MRI (r=0.54 to 0.67, p<0.0001) and clinical (r=0.31 to 0.36, p<0.0001) variables than MPD (r=0.23 to 0.41, p<0.001 for MRI and r=0.13 to 0.18; p=0.006 to p<0.001 for clinical variables). The GMF and BPF showed a slightly stronger relationship with all clinical variables (r=0.33 to 0.48; p<0.0001), when compared to both lesion and DWI measures. ENT (R2=0.28; p<0.0001) and GMF (R2=0.26; p<0.001) were best related with SP disease course. This study highlights the validity of DWI in discerning differences between NC and MS patients, as well as between different MS subtypes. ENT is a sensitive marker of overall brain damage that is strongly related to clinical impairment in patients with SP MS.

Dynamics of interferon-beta modulated mRNA biomarkers in multiple sclerosis patients with anti-interferon-beta neutralizing antibodies.

The objective of this study was to evaluate multiple interferon (IFN) specific mRNA biomarkers in multiple sclerosis (MS) patients with anti-IFN-beta neutralizing antibodies (NAB) using a pharmacodynamic study design. Thirty patients were enrolled. Blood samples were drawn at pre-treatment, 4-, 8-h time points following the intramuscular dose of IFN-beta-1a. Total RNA was obtained from peripheral blood cells, processed to cDNA and analyzed using quantitative real-time polymerase chain reaction. Pre-treatment serum samples were analyzed for anti-IFN-beta binding and neutralizing antibodies: 22 patients were NAB negative; equal numbers of the eight remaining patients were either NAB positive or had borderline NAB status. The results showed that early assessment (at 4 h after IFN-beta injection) of mRNAs for Stat-1, MxA, MxB and TRAIL was more sensitive than the later measurements. Furthermore, the NAB positive patients had strongly attenuated gene expression responses on all the mRNAs. Patients with borderline NAB had average responses that appear to be lower than NAB negative patients on several genes, notably Stat-1, TRAIL and beta2 microglobulin.

Reliable screening for neuropsychological impairment in multiple sclerosis.

In an earlier study, we developed the Multiple Sclerosis Neuropsychological Screening Questionnaire (MSNQ) to assist in the screening for neuropsychological (NP) impairments. Self-report MSNQ scores correlated significantly with measures of depression, whereas informant-report MSNQ scores correlated with cognitive performance, but not depression. This study was criticized for use of a small sample and lack of data regarding normal performance and test-retest reliability. The present study was designed to replicate the earlier work with a larger sample of patients and normal controls obtained from multiple sites. We also evaluated the test-retest reliability and predictive validity of the MSNQ. The sample included 85 multiple sclerosis (MS) patients and 40 normal controls, matched on demographic variables. All participants completed the MSNQ and underwent NP testing. Thirty-four patients were re-examined at one week. Pearson and ANOVA techniques were utilized for univariate comparisons. Bayesian statistics were calculated to assess predictive validity. Patient self- and informant-report MSNQ scores differed from normal and test retest reliability indices were high. Both self- and informant-reports were correlated with cognitive dysfunction and depression scales. Self-report MSNQ scores correlated more strongly with depression than cognitive performance, whereas the opposite pattern was observed with informant-report scores. Bayesian statistics showed that informant-report MSNQ scores predict cognitive impairment and patient self-report scores identify patients with cognitive impairment or depression. It is concluded that the MSNQ is useful, although patient self-reports may be exaggerated in depressed patients or reduced in patients with severe cognitive impairment.