ß2 glycoprotein I participates in phagocytosis of apoptotic neurons and in vascular injury in experimental brain stroke.

Beta-2 Glycoprotein I (ß2-GPI) is the main target of anti-phospholipid antibodies (aPL) in the autoimmune anti-phospholipid syndrome, characterized by increased risk of stroke. We here investigated the antibody independent role of ß2-GPI after ischemia/reperfusion, modeled in vivo by transient middle cerebral artery occlusion (tMCAo) in male C57Bl/6J mice; in vitro by subjecting immortalized human brain microvascular endothelial cells (ihBMEC) to 16 h hypoxia and 4 h re-oxygenation. ApoH (coding for ß2-GPI) was upregulated selectively in the liver at 48 h after tMCAo. At the same time ß2-GPI circulating levels increased. ß2-GPI was detectable in brain parenchyma and endothelium at all time points after tMCAo. Parenchymal ß2-GPI recognized apoptotic neurons (positive for annexin V, C3 and TUNEL) cleared by CD68+ brain macrophages. Hypoxic ihBMEC showed increased release of IL-6, over-expression of thrombomodulin and IL-1α after re-oxygenation with ß2-GPI alone. ß2-GPI interacted with mannose-binding lectin in mouse plasma and ihBMEC medium, potentially involved in formation of thrombi. We show for the first time that brain ischemia triggers the hepatic production of ß2-GPI. ß2-GPI is present in the ischemic endothelium, enhancing vascular inflammation, and extravasates binding stressed neurons before their clearance by phagocytosis. Thus ß2-GPI may be a new mediator of brain injury following ischemic stroke.

Free Thiol ß2-GPI (ß-2-Glycoprotein-I) Provides a Link Between Inflammation and Oxidative Stress in Atherosclerotic Coronary Artery Disease.

OBJECTIVE: Atherosclerotic coronary artery disease is well recognised as an inflammatory disorder that is also influenced by oxidative stress. ß2-GPI (ß-2-glycoprotein-I) is a circulating plasma protein that undergoes post-translational modification and exists in free thiol as well as oxidized forms. The aim of this study was to assess the association between these 2 post-translational redox forms of ß2-GPI and atherosclerotic coronary artery disease. Approach and Results: Stable patients presenting for elective coronary angiography or CT coronary angiography were prospectively recruited. A separate group of patients after reperfused ST-segment-elevation myocardial infarction formed an acute coronary syndrome subgroup. All patients had collection of fasting serum and plasma for quantification of total and free thiol ß2-GPI. Coronary artery disease extent was quantified by the Syntax and Gensini scores. A total of 552 patients with stable disease and 44 with acute coronary syndrome were recruited. While total ß2-GPI was not associated with stable coronary artery disease, a higher free thiol ß2-GPI was associated with its presence and extent. This finding remained significant after correcting for confounding variables, and free thiol ß2-GPI was a better predictor of stable coronary artery disease than hs-CRP (high-sensitivity C-reactive protein). Paradoxically, there were lower levels of free thiol ß2-GPI after ST-segment-elevation myocardial infarction. CONCLUSIONS: Free thiol ß2-GPI is a predictor of coronary artery disease presence and extent in stable patients. Free thiol ß2-GPI was a better predictor than high-sensitivity C-reactive protein.

Antiphospholipid antibodies in patients with lupus nephritis: clinical correlations and associations with long-term outcomes.

Whether the presence or absence of antiphospholipid antibodies (aPL) in patients with lupus nephritis (LN) is associated with differences in clinical outcomes remains unclear. We reviewed LN patients at a single centre during 2000-2017, and compared the clinical features and long-term outcomes between patients who were seropositive or seronegative for aPL. aPL was detected in 53/149 (35.6%) patients with biopsy-proven LN, and anticardiolipin IgM, anticardiolipin IgG, anti-ß2 glycoprotein I and lupus anticoagulant was detected in 18.8%, 18.1%, 10.7% and 8.1%, respectively. Follow-up was 155.8 ± 61.0 months, and was similar between aPL-seropositive and -seronegative patients. aPL seropositivity persisted in 94.3% of patients during remission. aPL-seropositive patients showed inferior patient survival (91% and 85% at 10 and 15 years, respectively, compared to 99% and 95% in aPL-seronegative patients; p = 0.043). Nine (6.0%) patients died during follow-up, including six aPL-seropositive (four thrombotic events and two bleeding complications related to anticoagulation) and three aPL-seronegative patients. aPL seropositivity was associated with more rapid decline in estimated glomerular filtration rate (-1.44 mL/min/year compared to -0.38 mL/min/year in aPL-seronegative patients; p = 0.027) and inferior long-term renal survival (82% and 74% at 10 and 15 years, respectively, compared to 91% and 87% in aPL-seronegative patients; p = 0.034). aPL-seropositive patients also had a higher incidence of thrombotic events and miscarriage (32.1% and 13.2%, respectively, compared to 16.7% and 2.1% in the aPL-seronegative group; p = 0.030 and 0.006). We concluded that aPL seropositivity was associated with inferior long-term patient and renal survival and more frequent thrombotic events and miscarriage in LN patients.

Reduced ß2-GPI is associated with increased platelet aggregation and activation in patients with prolonged isolated thrombocytopenia after allo-HSCT.

We aimed to measure platelet function and its relationship with ß2-GPI in prolonged isolated thrombocytopenia (PT) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Fifty-six patients with PT and 60 allo-HSCT recipients without PT (non-PT controls) were enrolled. Platelet aggregation and activation, ß2-GPI and anti-ß2-GPI antibody levels, vWF antigen, and vWF activity were analyzed. The effect of ß2-GPI on platelet aggregation was also measured ex vivo. Results showed that ADP-induced platelet aggregation significantly increased (39%±7.5% vs. 23%±8.5%, P=0.032), and the platelet expression of both CD62p (33.6%±11.6% vs. 8.5%±3.5%, P<0.001) and PAC-1 (42.4%±7.6% vs. 6.8%±2.2%, P<0.001) was significantly higher in patients with PT than in those without PT. Significantly lower ß2-GPI levels (164.2±12 µg mL-1 vs. 234.2±16 µg mL-1, P<0.001), higher anti-ß2-GPI IgG levels (1.78±0.46 U mL-1 vs. 0.94±0.39 U mL-1, P<0.001), and increased vWF activity (133.06%±30.50% vs. 102.17%±25.90%, P<0.001) were observed in patients with PT than in those without PT. Both ADP-induced platelet aggregation (n=116, r2=-0.5042, P<0.001) and vWF activity (n=116, r2=-0.2872, P<0.001) were negatively correlated with ß2-GPI levels. In summary, our data suggested that platelet aggregation and activation were significantly higher in patients with PT than in those without PT, which might be associated with reduced ß2-GPI levels. The reduced ß2-GPI levels might be due to the existence of anti-ß2-GPI IgG.

Predictive Value of Oxidized Low-Density Lipoprotein/ß2-Glycoprotein-I Complexes (oxLDL/ß2GPI) in Nonautoimmune Atherothrombosis.

INTRODUCTION: Lipid oxidation is a definite feature of atherosclerosis, and oxidized low-density lipoprotein (oxLDL) is not only highly immunogenic but toxic to several cell types. Beta-2-glycoprotein-I (ß2GPI) dampens oxLDL toxicity by forming binary oxLDL/ß2GPI complexes. We evaluated whether circulating oxLDL/ß2GPI complexes are associated to atherosclerosis-related events (ARE) and to venous thromboembolism (VTE). METHODS: In a cross-sectional case-control study, cases were (a) 57 consecutive patients (male/female [M/F] 33/24, mean age 57 [10] years) attending a thrombosis unit for ARE (myocardial infarction [MI] n = 20, peripheral vascular disease n = 7, and ischemic strokes n = 30); (b) 52 consecutive patients (M/F 22/30, mean age 55 [17] years) attending the same unit for unprovoked (VTE); (c) normal controls comprised 90 participants (M/F 35/55, mean age 41 [15] years); and (d) oxLDL/ß2GPI complexes were measured by immunoassay and resulting levels divided into quartiles. RESULTS: The odds ratio (OR) of ARE was greater in the fourth and second quartiles than in the first quartile (8.5 and 6.0, respectively); the OR of developing MI was greatest in the fourth quartile (17.8). By multivariable analysis with age, sex, smoking, lipid status, statin, and ARE phenotypes as independent variables and oxLDL/ß2GPI as the dependent variable, only MI predicted oxLDL/ß2GPI ( P < .0001). CONCLUSIONS: OxLDL/ß2GPI may be regarded as a marker of ARE, in particular of MI.

Anti-phospholipid syndrome in seven leprosy patients with thrombotic events on corticosteroid and/or thalidomide regimen: insights on genetic and laboratory profiles

Abstract INTRODUCTION Corticosteroids and/or thalidomides have been associated with thromboembolism events (TBE) in multibacillary (MB) leprosy. This report aimed to determine genetic and laboratory profiles associated with leprosy and TBE. METHODS Antiphospholipid antibodies (aPL), coagulation-related exams, prothrombin and Leiden's factor V mutations, and ß2-glycoprotein-I (ß2GPI) Val247Leu polymorphism were assessed. RESULTS Six out of seven patients with leprosy were treated with prednisone and/or thalidomide during TBE and presented at least one positive aPL. All patients presented ß2GPI polymorphism, and one showed prothrombin mutation. CONCLUSIONS Corticosteroid or thalidomide adverse effects and aPL and ß2GPI polymorphisms may cause TBE in patients with MB leprosy.

ß2-Glycoprotein I/IgA Immune Complexes: A Marker to Predict Thrombosis After Renal Transplantation in Patients With Antiphospholipid Antibodies.

BACKGROUND: Antiphospholipid syndrome is characterized by recurrent thrombosis and gestational morbidity in patients with antiphospholipid autoantibodies (aPLs). Predictive value of the presence of aPLs is low, and new markers are necessary to identify aPL carriers at higher risk and take preventive measures on them. The presence of circulating immune complexes of IgA bound to ß2-glycoprotein I (B2A-CIC) has been associated with occurrence of acute thrombotic events. In this work we study its possible predictive value for the appearance of acute thrombotic events in patients who are going to undergo transplant surgery, a well-known trigger of acute thrombotic events in aPL carriers. METHODS: We performed a follow-up study based on the Magnum 12+12 Cohort of patients who received a kidney transplant (n=1339). Three groups were established: group 1 patients who were positive for IgA anti-ß2-glycoprotein I (aB2GP1) and B2A-CIC (n=125); group 2 patients who were positive only for IgA aB2GP1 (n=240); and control group, patients who were negative for IgA aB2GP1 (n=974). Levels of autoantibodies and B2A-CIC were quantified immediately before the transplant surgery and patients were followed up for 6 months. RESULTS: In group 1, 46.4% of patients experienced any type of thrombosis versus 10.4% in group 2 (P<0.001) and 8.6% in the control group (P<0.001). The incidence of graft thrombosis in group 1 (31.2%) was significantly higher than that observed in group 2 (3.3%, P<0.001) and the control group (2.6%, P<0.001). In a multivariate analysis, the presence of B2A-CIC was an independent variable to experience any type of posttransplant thrombosis (hazard ratio, 6.72; 95% confidence interval, 4.81-9.37) and, prominently, for graft thrombosis (hazard ratio, 14.75; 95% confidence interval, 9.11-23.89). No significant differences were found between B2A-CIC-negative and control group patients. CONCLUSIONS: The presence of B2A-CIC is a predictor of acute thrombotic events. Patients who were positive for IgA aB2GP1 only are at risk of experiencing thrombosis if they are B2A-CIC positive. If they are B2A-CIC-negative patients, they have the same risk as the control group. Treatments to prevent acute thrombotic events should focus on B2A-CIC-positive patients.

Impact of adalimumab treatment on cardiovascular risk biomarkers in psoriasis: Results of a pilot study.

Psoriasis is a chronic systemic immune-mediated inflammatory dermatosis associated with several comorbidities. Psoriasis patients are at increased risk of developing cardiovascular diseases (CVD), namely, coronary heart disease, stroke or peripheral vascular disease, and psoriasis seems to be an independent cardiovascular risk factor. Antipsoriatic systemic therapy, especially anti-tumor necrosis factor (TNF)-α, seems to exert a beneficial effect on these comorbidities. The purpose of this study was: (i) to measure the level of cardiovascular serum markers in psoriasis patients in comparison with healthy volunteers; and (ii) to compare the serum level of the same markers in patients before and 3 months after adalimumab therapy. We investigated six biomarkers connected to CVD: C-reactive protein (measured high sensitively, hsCRP), oxidized low-density lipoproteins (oxLDL), oxLDL/ß-glycoprotein I complex (oxLDL/ß2GPI), vascular endothelial adhesion molecule 1 (VCAM-1), E-selectin and interleukin (IL)-22. These biomarkers were measured in 21 patients with moderate/severe psoriasis before and after treatment with adalimumab and in healthy volunteers. hsCRP (P < 0.05), oxLDL-ß2GPI complex (P < 0.05), E-selectin (P < 0.001) and IL-22 (P < 0.001) were significantly increased in comparison with healthy controls, whereas oxLDL and VCAM-1 were also higher in psoriasis patients but the difference did not reach statistical significance. A decrease of E-selectin (P < 0.001) and IL-22 (P < 0.001) was observed after 3 months of adalimumab therapy. Inhibition of TNF-α seems to not only improve psoriasis but also decreases serum cardiovascular biomarkers. E-selectin and IL-22 could serve for monitoring of the efficacy of antipsoriatic systemic therapy on cardiovascular risk.

Thrombotic microangiopathy and poor renal outcome in lupus patients with or without antiphospholipid syndrome.

OBJECTIVES: To assess the presence of acute thrombotic microangiopathy (aTMA) and chronic vascular lesions (cTMA) in lupus nephropathy, and to evaluate their association with extrarrenal lupus features, aPL positivity, antiphospholipid syndrome (APS) and renal survival. METHODS: We studied lupus patients with renal biopsy, ≥1 year of post-biopsy follow-up and at least two aCL (IgG-IgM), anti-ß2GP-I (IgG-IgM) and/or lupus anticoagulant (LAC) determinations. A blinded nephropathologist evaluated all biopsies. We retrospectively collected clinical, serological, treatment and renal survival data. We plotted survival curves and used Cox regression analysis. RESULTS: A total of 90 biopsies were included with a median disease duration 5.9 years and median follow-up 2.4 years. Eleven patients (12.2%) had cTMA and 3 (3%) aTMA. There was no difference in age, lupus duration, hypertension, drugs, APS, non-renal lupus features, low C3 or C4 aCL IgG, anti-ß2GP1-IgG or IgM and LAC between cTMA and non-cTMA groups. The cTMA group had aCL-IgM less frequently (27% vs. 66%, p=0.02), more class IV nephropathy (100% vs. 40%, p=0.01), higher activity index scores (7.5 vs. 2, p=0.03) and a tendency to need chronic dialysis (54.5% vs. 24% p=0.06). At four years of follow-up, 28% of the cTMA group and 62% of the non-cTMA group were free of dialysis (log rank p=0.03). cTMA was associated with chronic dialysis (RR 2.9, CI 95% 1.1-8.1, p=0.03). CONCLUSIONS: cTMA conferred a poor renal outcome. We found a low frequency of TMA that was not associated with with APL positivity or APS, suggesting that other factors hitherto not studied are involved in its pathogenesis.

A contribution to detection of anticardiolipin and anti-ß2glycoprotein I antibodies: Comparison between a home-made ELISA and a fluorescence enzyme immunoassay.

BACKGROUND: Currently, ELISA for detection of anticardiolipin (aCL) and anti-ß2glycoprotein I (anti-ß2GPI) antibodies is not standardized. Recently, few studies have compared the performance of ELISA with that of fluorescence enzyme immunoassay (FEIA), but they have produced debatable results. The aim of this investigation was to compare ELISA with FEIA results in detecting aCL and anti-ß2GPI antibodies. METHODS: The study cohort included 94 primary antiphospholipid syndrome (PAPS) patients, 65 subjects with the clinical criteria for PAPS classification but ELISA negative for the laboratory criteria and 165 control subjects. Serum IgG/IgM aCL/anti-ß2GPI antibodies were determined using FEIA-EliA™ and a home-made ELISA. RESULTS: The sensitivities of the two methods were similar with the exception of IgM aCL which was found to be significantly higher in the PAPS patients using the ELISA method, even if IgM aCL was detected at a low level by both techniques. The two assays had a comparable specificity, a high/significant agreement and a significant correlation between the antibody levels. FEIA testing uncovered no significant prevalence of any antiphospholipid (aPL) antibody in the ELISA negative patients. CONCLUSION: Our results suggest that FEIA is comparable to a home-made ELISA.

Improvement of Plasma Biomarkers after Switching Stroke Patients from Other Angiotensin II Type I Receptor Blockers to Olmesartan.

BACKGROUND: Managing hypertension is crucial for preventing stroke recurrence. Some stroke patients experience resistant hypertension. In our experimental stroke model, olmesartan increased the expression of angiotensin (Ang) II converting enzyme-2. We hypothesized that switching to olmesartan affects biomarkers and the blood pressure (BP) in stroke patients whose BP is insufficiently controlled by standard doses of Ang II type I receptor blockers (ARBs) other than olmesartan. METHODS: We recruited 25 patients to study our hypothesis. All had a history of stroke or silent cerebral infarction. We switched them to olmesartan (10-40 mg per day) for 12 weeks and determined their plasma level of Ang-(1-7), peroxiredoxin, oxidized low-density lipoprotein (oxLDL)/ß-2-glycoprotein I (ß2GPI) complex, adiponectin, high mobility group box 1 (HMGB1), and tumor necrosis factor-α (TNFα) and recorded their BP before and after olmesartan treatment. RESULTS: After switching the patients to olmesartan, their plasma level of Ang-(1-7) as a vasoprotective indicator and adiponectin regulating metabolic syndrome was increased, and peroxiredoxin and the oxLDL/ß2GPI complex indicating its antioxidative stress and its proatherogenicity were lower than their baseline. This suggests that olmesartan may be more effective than other ARBs to improve these conditions. Neither HMGB1 nor TNFα reflecting an inflammatory response was affected, suggesting that the anti-inflammatory effects of olmesartan are similar to those of other ARBs. The recommended BP (<140/90) was obtained in 10 of the 25 patients after switching to olmesartan. No adverse events occurred. CONCLUSIONS: Switching from other ARBs to olmesartan may be a promising therapeutic option in patients with resistant hypertension.

Screening and identification of potential predictive biomarkers for Down's syndrome from second trimester maternal serum.

OBJECTIVE: In this study, we aimed to search for noninvasive predictive biomarkers for prenatal diagnosis of Down's syndrome (DS). METHODS: Maternal serum samples from five DS-affected pregnant women and five DS-unaffected women were analyzed by 2D gel electrophoresis and MALDI-TOF mass spectrometry to screen for potential predictive biomarkers of DS. Then, differential levels of dGTPase, ß2-glycoprotein I (ß2-GPI), complement factor H-related protein 1 precursor (CFHR1) and kininogen 1 isoform 2 were further verified by western blotting tests in another independent group. RESULTS: Statistical analysis results revealed 29 protein spots whose levels differed significantly in the DS-affected pregnancies group. Of these, the eight most differentially expressed in DP were identified successfully. Among these, levels of dGTPase, CFHR1 and kininogen 1 were elevated significantly, whereas ß2-GPI was reduced in DP. DISCUSSION: These preliminarily verified proteins might serve as potential predictive biomarkers for DS-affected pregnancies.

Novel serum protein biomarker panel revealed by mass spectrometry and its prognostic value in breast cancer.

INTRODUCTION: Serum profiling using proteomic techniques has great potential to detect biomarkers that might improve diagnosis and predict outcome for breast cancer patients (BC). This study used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) to identify differentially expressed proteins in sera from BC and healthy volunteers (HV), with the goal of developing a new prognostic biomarker panel. METHODS: Training set serum samples from 99 BC and 51 HV subjects were applied to four adsorptive chip surfaces (anion-exchange, cation-exchange, hydrophobic, and metal affinity) and analyzed by time-of-flight MS. For validation, 100 independent BC serum samples and 70 HV samples were analyzed similarly. Cluster analysis of protein spectra was performed to identify protein patterns related to BC and HV groups. Univariate and multivariate statistical analyses were used to develop a protein panel to distinguish breast cancer sera from healthy sera, and its prognostic potential was evaluated. RESULTS: From 51 protein peaks that were significantly up- or downregulated in BC patients by univariate analysis, binary logistic regression yielded five protein peaks that together classified BC and HV with a receiver operating characteristic (ROC) area-under-the-curve value of 0.961. Validation on an independent patient cohort confirmed the five-protein parameter (ROC value 0.939). The five-protein parameter showed positive association with large tumor size (P = 0.018) and lymph node involvement (P = 0.016). By matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS, immunoprecipitation and western blotting the proteins were identified as a fragment of apolipoprotein H (ApoH), ApoCI, complement C3a, transthyretin, and ApoAI. Kaplan-Meier analysis on 181 subjects after median follow-up of >5 years demonstrated that the panel significantly predicted disease-free survival (P = 0.005), its efficacy apparently greater in women with estrogen receptor (ER)-negative tumors (n = 50, P = 0.003) compared to ER-positive (n = 131, P = 0.161), although the influence of ER status needs to be confirmed after longer follow-up. CONCLUSIONS: Protein mass profiling by MS has revealed five serum proteins which, in combination, can distinguish between serum from women with breast cancer and healthy control subjects with high sensitivity and specificity. The five-protein panel significantly predicts recurrence-free survival in women with ER-negative tumors and may have value in the management of these patients.

Identification of serum proteome components associated with progression of non-small cell lung cancer.

The aim of the present study was to perform comparative analysis of serum from patients with different stages of non-small cell lung cancer (NSCLC) using the three complementary proteomic approaches to identify proteome components associated with the progression of cancer. Serum samples were collected before any treatment from 200 patients with NSCLC, including 103 early stage, 64 locally advanced and 33 metastatic cancer samples, and from 200 donors without malignancy. The low-molecular-weight fraction of serum proteome was MALDI-profiled in all samples. Serum proteins were characterized using 2D-PAGE and LC-MS/MS approaches in a representative group of 30 donors. Several significant differences were detected between serum samples collected from patients with early stage cancer and patients with locally advanced cancer, as well as between patients with metastatic cancer and patients with local disease. Of note, serum components discriminating samples from early stage cancer and healthy persons were also detected. In general, about 70 differentiating serum proteins were identified, including inflammatory and acute phase proteins already reported to be associated with the progression of lung cancer (serum amyloid A or haptoglobin). Several differentiating proteins, including apolipoprotein H or apolipoprotein A1, were not previously associated with NSCLC. No significant differences in patterns of serum proteome components were detected between patients with adenocarcinoma and squamous cell carcinoma. In conclusion, we identified the biomarker candidates with potential importance for molecular proteomic staging of NSCLC. Additionally, several serum proteome components revealed their potential applicability in early detection of the lung cancer.

Antiphospholipid antibodies in critically ill patients with cancer: a prospective cohort study.

PURPOSE: The purpose of this study is to evaluate the prevalence and the prognostic impact of antiphospholipid antibodies (aPL) in critically ill cancer patients. METHODS: This is a prospective cohort study in adult patients admitted to the intensive care unit for more than 48 hours at a cancer center. Clinical and laboratory data including coagulation parameters were obtained. Cox proportional hazard models were used to identify predictors of 6-month mortality. RESULTS: Ninety-five (solid tumor, 79%; hematologic malignancies, 21%) patients were included, and aPL were identified in 74% of them. Median Simplified Acute Physiology Score 3 and Sequential Organ Failure Assessment scores were 51 (37-65) and 5 (2-8) points, respectively. The most frequent aPL were lupus anticoagulant (61%) and anti-ß2 glicoprotein I (32%). Vascular complications occurred in 18% of patients and were comparable between aPL+ and aPL- patients. Sepsis and need for renal replacement therapy were more frequent in aPL+ patients. Hospital and 6-month mortality rates were 44% and 56%, respectively. Higher Sequential Organ Failure Assessment scores (each point) (hazard ratios [HR]=2.83 [95% confidence interval, 1.59-5.00]), medical admissions (HR=2.66 [1.34-5.27]), and d-dimer more than 500 ng/dL (HR=1.89 (1.04-3.44]) were independently associated with mortality. After adjusting for these covariates, aPL status was not associated with outcomes (HR=1.22 [0.60-2.47]). CONCLUSIONS: Lupus anticoagulants were frequent in critically ill cancer patients. However, they were not associated with medium-term survival in these patients.

Serum protein profile of Crohn's disease treated with infliximab.

The infliximab (IFX) has dramatically improved the treatment of Crohn's disease (CD). However, the need for predictive factors, indicative of patients' response to IFX, has yet to be met. In the current study, proteomics technologies were employed in order to monitor for differences in protein expression in a cohort of patients following IFX administration, aiming at identifying a panel of candidate protein biomarkers of CD, symptomatic of response to treatment. We enrolled 18 patients, who either had achieved clinical and serological remission (Rm, n=6), or response (Rs, n=6) and/or were PNRs (n=6), to IFX. Serum samples were subjected to two-dimensional Gel Electrophoresis. Following evaluation of densitometrical data, protein spots exhibiting differential expression among the groups, were further characterized by MALDI-TOF-MS. Identified proteins where evaluated by immunoblot analysis while functional network association was carried out to asses significance. Proteins apolipoprotein A-I (APOA1), apolipoprotein E (APOE), complement C4-B (CO4B), plasminogen (PLMN), serotransferrin (TRFE), beta-2-glycoprotein 1 (APOH), and clusterin (CLUS) were found to be up-regulated in the PNR and Rs groups whereas their levels displayed no changes in the Rm group when compared to baseline samples. Additionally, leucine-rich alpha-2-glycoprotein (A2GL), vitamin D-binding protein (VTDB), alpha-1B-glycoprotein (A1BG) and complement C1r subcomponent (C1R) were significantly increased in the serum of the Rm group. Through the incorporation of proteomics technologies, novel serum marker-molecules demonstrating high sensitivity and specificity are introduced, hence offering an innovative approach regarding the evaluation of CD patients' response to IFX therapy.

Genetic determinants of plasma ß2-glycoprotein I levels: a genome-wide association study in extended pedigrees from Spain.

BACKGROUND: ß2 -Glycoprotein I (ß2 -GPI), also designated apolipoprotein H, is a 50-kDa protein that circulates in blood at high concentrations, playing important roles in autoimmune diseases, hemostasis, atherogenesis, and angiogenesis, as well as in host defense against bacteria and in protein/cellular waste removal. Plasma ß2 -GPI levels have a significant genetic component (heritability of ~ 80%). OBJECTIVES: To present the results of a genome-wide association study for plasma ß2 -GPI levels in a set of extended pedigrees from the Genetic Analysis of Idiopathic Thrombophilia (GAIT) Project. PATIENTS/METHODS: A total of 306 individuals for whom ß2 -GPI plasma measurements were available were typed for 307,984 single-nucleotide polymorphisms (SNPs) with the Infinium 317k Beadchip (Illumina). Association with the ß2 -GPI phenotype was investigated through variance component analysis, and the most significant results were followed up for association with coronary artery disease (CAD) in an independent in silico analysis involving 5765 CAD cases from the PROCARDIS Project and 7264 controls from the PROCARDIS Project and the Wellcome Trust Case Control Consortium (WTCCC) collection. RESULTS: After correction for multiple testing, three SNPs located in/around two genes (ELF5 and SCUBE2) reached genome-wide significance. Moreover, an SNP in the APOH gene showed suggestive association with the ß2 -GPI phenotype. Some of the identified genes are plausible biological candidates, as they are actually or potentially involved in inflammatory processes. CONCLUSIONS: Our results represent a first step towards identifying common variants reflecting the genetic architecture influencing plasma ß2 -GPI levels, and warrant further validation by functional experiments, as the functions of some of the discovered loci are still unknown.

Use of MDLC-DIGE and LC-MS/MS to identify serum biomarkers for complete remission in patients with acute myeloid leukemia.

The response criteria for complete remission (CR) in acute myeloid leukemia (AML) are currently based on morphology and blood cell counts. However, these criteria are insufficient to establish a diagnosis in cases with poor quality bone marrow (BM) samples demonstrating a loss of cellular morphology. We investigated whether the sera of patients contained biomarkers that indicate disease response status. First, we performed multidimensional liquid chromatography-differential gel electrophoresis (MDLC-DIGE) to generate protein profiles of two pooled, paired serum samples from patients who had achieved CR; one collected at diagnosis (PreCR) and the other collected after chemotherapy (CR). Then, with the biomarker candidates found, ELISA was carried out for individual PreCR and CR samples, and for other verification sets including nonremission (NR) patients and normal samples. We selected two proteins, complement factor H (CFH) and apolipoprotein H (ApoH), with dye (Cy) ratios showing greater than 2.0-fold differences between the pooled samples. ELISA showed that CFH and ApoH are useful for distinguishing between the recovered (CR and normal) and nonrecovered (PreCR, PreNR, and NR) states in AML (p <0.001). We successfully applied a protein profiling technology of MDLC-DIGE and LC-MS/MS to discover two biomarkers for CR which needs further validation for a clinical setting.

Nutritional vitamin D supplementation in haemodialysis: A potential vascular benefit?

AIM: Vitamin D deficiency is highly prevalent in end-stage renal disease and has been associated with atherosclerosis, endothelial dysfunction and left ventricular hypertrophy. Although activated vitamin D has shown to be cardioprotective, the cardiovascular benefits of nutritional vitamin D (i.e. ergocalciferol or cholecalciferol) have not been explored in the dialysis population. The aim of this investigation was to evaluate the effect of ergocalciferol therapy on vascular adhesion molecules, markers of inflammation and atherosclerosis among haemodialysis patients. METHODS: This was a pilot study of matched haemodialysis patients. For every patient enrolled taking ergocalciferol, an age and race matched control was recruited. Predialysis blood samples were collected and assayed for adhesion molecules (soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1), E-selectin and P-selectin), inflammatory cytokines (interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α)), oxLDL-ß(2) GPI and IgG anticardiolipin. RESULTS: A total of 40 haemodialysis patients were studied (20 on ergocalciferol therapy, 20 not receiving ergocalciferol therapy). Patients taking ergocalciferol had higher 25-hydroxyvitamin D levels compared with those not taking ergocalciferol. Even though doxercalciferol usage and dosing was similar between groups, plasma sVCAM-1, sICAM-1 and P-selectin concentrations were lower among ergocalciferol treated patients. No significant differences in E-selectin, IL-6, TNF-α, oxLDL-ß(2) GPI or anticardiolipin antibody levels were observed. CONCLUSION: Patients receiving ergocalciferol had lower plasma levels of vascular adhesion molecules despite equivalent use of activated vitamin D therapy. Future investigations should confirm the role of nutritional vitamin D therapy, in addition to activated D therapy, in haemodialysis patients and the potential vascular benefits of these agents.

ß2-glycoprotein I: a novel component of innate immunity.

Sepsis is a systemic host response to invasive infection by bacteria. Despite treatment with antibiotics, current mortality rates are in the range of 20%-25%, which makes sepsis the most important cause of death in intensive care. Gram-negative bacteria are a prominent cause of sepsis. Lipopolysaccharide (LPS), one of the major constituents of the outer membrane of Gram-negative bacteria, plays a major role in activating the host's immune response by binding to monocytes and other cells. Several proteins are involved in neutralization and clearance of LPS from the bloodstream. Here, we provide evidence that ß2-glycoprotein I (ß2GPI) is a scavenger of LPS. In vitro, ß2GPI inhibited LPS-induced expression of tissue factor and IL-6 from monocytes and endothelial cells. Binding of ß2GPI to LPS caused a conformational change in ß2GPI that led to binding of the ß2GPI-LPS complex to monocytes and ultimately clearance of this complex. Furthermore, plasma levels of ß2GPI were inversely correlated with temperature rise and the response of inflammatory markers after a bolus injection of LPS in healthy individuals. Together, these observations provide evidence that ß2GPI is involved in the neutralization and clearance of LPS and identify ß2GPI as a component of innate immunity.