Vitamin C, Hydrocortisone, and the Combination Thereof Significantly Inhibited Two of Nine Inflammatory Markers Induced by Escherichia Coli But Not by Staphylococcus Aureus - When Incubated in Human Whole Blood.

ABSTRACT: Vitamin C combined with hydrocortisone is increasingly being used to treat septic patients, even though this treatment regimen is based on questionable evidence. When used, a marked effect on key players of innate immunity would be expected, as sepsis is featured by a dysregulated immune response.Here, we explored the effect of vitamin C and hydrocortisone alone and combined, in an ex vivo human whole-blood model of Escherichia coli- or Staphylococcus aureus-induced inflammation. Inflammatory markers for activation of complement (terminal C5b-9 complement complex [TCC]), granulocytes (myeloperoxidase), platelets (ß-thromboglobulin), cytokines (tumor necrosis factor [TNF], IL-1ß, IL6, and IL-8), and leukocytes (CD11b and oxidative burst) were quantified, by enzyme-linked immunosorbent assay, multiplex technology, and flow cytometry.In E. coli- and S. aureus-stimulated whole blood, a broad dose-titration of vitamin C and hydrocortisone alone did not lead to dose-response effects for the central innate immune mediators TCC and IL-6. Hence, the clinically relevant doses were used further. Compared to the untreated control sample, two of the nine biomarkers induced by E. coli were reduced by hydrocortisone and/or vitamin C. TNF was reduced by hydrocortisone alone (19%, P = 0.01) and by the combination (31%, P = 0.01). The oxidative burst of monocytes and granulocytes was reduced for both drugs alone and their combination, (ranging 8-19%, P < 0.05). Using S. aureus, neither of the drugs, alone nor in combination, had any effects on the nine biomarkers.In conclusion, despite the limitation of the ex vivo model, the effect of vitamin C and hydrocortisone on bacteria-induced inflammatory response in human whole blood is limited and following the clinical data.

Increased CCL24 and CXCL7 levels in the cerebrospinal fluid of patients with neurosyphilis.

BACKGROUND: Monocytes are recruited into the cerebrospinal fluid (CSF) of patients with neurosyphilis, suggesting abnormal chemokine expression. We aimed to investigate the aberrant expression of chemokines in the CSF of these patients. METHODS: CSF and serum samples were collected from patients with neurosyphilis between July 2017 and June 2019 in the Dermatology Department, Second Affiliated Hospital of Zhejiang University. Differences in the expression of 38 chemokines between patients with and without neurosyphilis were detected using RayBio® Human Chemokine Antibody Array C1. CCL24 and CXCL7 levels in the patients' CSF and serum were further measured using RayBio® CCL24 and CXCL7 ELISA kits. RESULTS: Ninety-three CSF and serum samples of patients with syphilis were collected. Antibody array analysis showed that the CSF levels of CCL24 (P = .0185), CXCL7 (P < .0001), CXCL13 (P < .0001), CXCL10 (P < .0001), and CXCL8 (P < .0001) were significantly higher in patients with than without neurosyphilis. ELISA confirmed significantly higher CCL24 and CXCL7 levels in the CSF of patients with than without neurosyphilis (CCL24: 6.082 ± 1.137 pg/mL vs 1.773 ± 0.4565 pg/mL, P = .0037; CXCL7: 664.3 ± 73.19 pg/mL vs 431.1 ± 90.54 pg/mL, P = .0118). Increased CCL24 and CXCL7 expression was seen throughout all neurosyphilis stages, had moderate diagnostic efficiency for neurosyphilis, and correlated poorly with CSF cell count and Venereal Disease Research Laboratory titer. CSF CCL24 levels also correlated poorly with CSF protein concentration. CONCLUSION: Abnormally high CSF chemokines levels may play a role in the pathogenesis of neurosyphilis.

Analysis by proteomics reveals unique circulatory proteins in idiopathic pulmonary fibrosis.

BACKGROUND AND OBJECTIVE: Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic disease that has a poor 3-year median survival rate with unclear pathophysiology. Radiological features include bibasal, subpleural fibrosis and honeycombing while its pathology is characterized by fibroblastic foci and honeycombing. Proteomic analysis of circulating molecules in plasma may identify factors that characterize IPF and may assist in the diagnosis, prognostication and determination of pathogenic pathways in this condition. METHODS: Two independent quantitative proteomic techniques were used, isobaric tags for relative and absolute quantitation (iTRAQ) and multiple reaction monitoring (MRM), to identify differentially expressed plasma proteins in a group of IPF patients in comparison to healthy controls with normal lung function matched for age and gender. RESULTS: Five proteins were identified to be differentially expressed in IPF compared to healthy controls (upregulation of platelet basic protein and downregulation of actin, cytoplasmic 2, antithrombin-III, extracellular matrix protein-1 and fibronectin). CONCLUSION: This study further validates the combinational use of non-targeted discovery proteomics (iTRAQ) with targeted quantitation by mass spectrometry (MRM) of soluble biomarkers to identify potentially important molecules and pathways for pulmonary diseases such as IPF.

Serum plasminogen as a potential biomarker for the effects of low-dose benzene exposure.

Exposure to low-dose benzene may lead to hematotoxicity and cause health problems. Though peripheral blood cell count is widely used in benzene exposure assessment and health risk assessment, the reports regarding the effects of low-dose benzene exposure on blood cell count remain inconsistent. To uncover more sensitive biomarkers for low-dose benzene exposure, our previous study screened out three potential serum proteins-plasminogen (PLG), platelet basic protein (PBP) and apolipoprotein B100 (ApoB100)-as biomarkers from chronic benzene poisoning patients by using proteomic analysis. In the present study, we verify the three serum proteins as biomarkers for the effects of low-dose benzene exposure in a large low-dose benzene exposure population. The study showed that serum PLG increased in benzene exposed workers and was positively correlated with benzene exposure levels. However, no significant changes in serum PBP or ApoB100 were found in the benzene exposed workers. To explore whether the candidate serum proteins are associated with hematotoxicity, the study population was regrouped into two groups, based on their WBC counts. Our results showed that the workers with high serum PLG levels suffered higher risk of WBC abnormalities than did workers with low serum PLG levels. Taken together, these findings indicate that the increase in serum PLG might be associated with low-dose benzene exposure and benzene-induced hematotoxicity. Thus, we suggest serum PLG could be used as a potential biomarker for the effects of low-dose benzene exposure.

Identification of candidate serum biomarkers of childhood-onset growth hormone deficiency using SWATH-MS and feature selection.

A typical clinical manifestation of growth hormone deficiency (GHD) is a short stature resulting from delayed growth, but GHD affects bone health, cardiovascular function and metabolic profile and therefore quality of life. Although early GH treatment during childhood has been shown to improve outcomes, no single biochemical parameter is currently available for the accurate diagnosis of GHD in children. There is hence a need for non-invasive biomarkers. In this study, the relative abundance of serum proteins from GHD children and healthy controls was measured by next-generation proteomics SWATH-MS technology. The data generated was analysed by machine-learning feature-selection algorithms in order to discover the minimum number of protein biomarkers that best discriminate between both groups. The analysis of serum proteins by a SWATH-MS approach yielded a useful method for discovering potential biomarkers of GHD in children. A total of 263 proteins were confidently detected and quantified in each sample. Pathway analysis indicated an effect on tissue/organ structure and morphogenesis. The top ten serum protein biomarker candidates were identified after applying feature-selection data analysis. The combination of three proteins - apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein - demonstrated the best classification performance for our data. In addition, the apolipoprotein group resulted in strong over-representation, thus highlighting these proteins as an additional promising biomarker panel. SIGNIFICANCE: Currently there is no single biochemical parameter available for the accurate diagnosis of growth hormone (GH) deficiency (GHD) in children. Simple GH measurements are not an option: because GH is released in a pulsatile action, its blood levels fluctuate throughout the day and remain nearly undetectable for most of that time. This makes measurements of GH in a single blood sample useless for assessing GH deficiency. Actually, the diagnosis of GHD includes a combination of direct and indirect non-accurate measurements, such as taking several body measurements, testing GH levels in multiple blood samples after provocative tests (GH peak <7.3ng/mL, using radioimmunoassay), and conducting magnetic resonance imaging (MRI), among others. Therefore, there is a need for simple, non-invasive, accurate and cost-effective biomarkers. Here we report a case-control study, where relative abundance of serum proteins were measured by next-generation proteomics SWATH-MS technology in 15 GHD children and 15healthy controls matched by age, sex, and not receiving any treatment. Data generated was analysed by machine learning feature selection algorithms. 263 proteins could be confidently detected and quantified on each sample. The top 10 serum protein biomarker candidates could be identified after applying a feature selection data analysis. The combination of three proteins, apolipoprotein A-IV, complement factor H-related protein 4 and platelet basic protein, showed the best classification performance for our data. In addition, the fact that the pathway and GO analysis we performed pointed to the apolipoproteins as over-represented highlights this protein group as an additional promising biomarker panel for the diagnosis of GHD and for treatment evaluation.

Platelet sequestration with a new-generation autotransfusion device.

BACKGROUND: Autologous platelet-rich plasma (PRP) can be used either to prevent platelets (PLTs) from lesions during extracorporeal circulation or for wound therapy, when processed into PLT gel. The aim of this study was to evaluate the PLT sequestration abilities of a new-generation autotransfusion device. STUDY DESIGN AND METHODS: In this experimental study the discontinuous, new-generation autotransfusion device XTRA was evaluated using fresh donor blood. The blood was processed in four different size bowls (X55, X125, X175, X225 [bowls' size in mL]) using the device's built-in "PLT sequestration program." PLT functionality was tested using aggregometry; for PLT activation, ß-thromboglobulin (ß-TG) and soluble P-selectin levels were determined. Cell damage was assessed by a morphology score and hypotonic shock response. Additionally, PLTs were tested after 6 hours to identify storage lesions. RESULTS: Platelet recovery in the PRP ranged from 39% to 64% and averaged 6.2-fold PLT enrichment as defined by the increase in PLT concentration. The preparation caused minimal cell damage and a decrease in cell function by only 10%, but a slight activation was observed amounting to 9% of the maximal ß-TG release. The efficiency of the preparation, represented by the PLT recovery rate, increased in a linear fashion with the increasing bowl sizes being tested. After 6 hours of storage the prepared PLTs showed an additional 9% loss in function, but only 4% decrease in viability. CONCLUSION: The autotransfusion device XTRA was capable of high-quality perioperative PRP preparation, and the bowl size was found to have an influence on the efficiency of the preparation.

CTAPIII/CXCL7: a novel biomarker for early diagnosis of lung cancer.

It is desirable to have a biomarker which can facilitate low-dose CT in diagnosis of early stage lung cancer. CTAPIII/CXCL7 is reported to be a potential biomarker for diagnosis of early lung cancer. In this study, we investigated the serum level of CTAPIII/CXCL7 in patients at different stage of lung cancer and the diagnostic efficacy of CTAPIII/CXCL7 in NSCLC. The plasma level of CTAPIII/CXCL7 was assayed by ELISA. CEA, SCCAg, and Cyfra211 were measured using a commercial chemiluminescent microparticle immunoassay. A total of 419 subjects were recruited, including 265 NSCLC patients and 154 healthy individuals. The subjects were randomly assigned to a training set and a test set. Receiver operating characteristic (ROC) and binary logistic regression analyses were conducted to evaluate the diagnostic efficacy and establish diagnostic mathematical model. Plasma CTAPIII/CXCL7 levels were significantly higher in NSCLC patients than in controls, which was independent of the stage of NSCLC. The diagnostic efficiency of CTAPIII/CXCL7 in NSCLC (training set: area under ROC curve (AUC) 0.806, 95% CI: 0.748-0.863; test set: AUC 0.773, 95% CI: 0.711-0.835) was greater than that of SCCAg, Cyfra21-1, or CEA. The model combining CTAPIII/CXCL7 with CEA, SCCAg, and Cyfra21-1 was more effective for NSCLC diagnosis than CTAPIII/CXCL7 alone. In addition, plasma level of CTAPIII/CXCL7 may contribute to the early diagnosis of NSCLC. CTAPIII/CXCL7 can be used as a plasma biomarker for the diagnosis of NSCLCs, particularly early stage lung cancer, with relatively high sensitivity and specificity.

Expression level of CXCL7 in peripheral blood cells is a potential biomarker for the diagnosis of renal cell carcinoma.

There are no blood biomarkers for the diagnosis of renal cell carcinoma (RCC) in routine clinical use. We focused on the gene expression profile of peripheral blood cells obtained from RCC patients to discover novel biomarkers for RCC diagnosis. Using microarray analysis and quantitative verification, CXCL7 was shown to be significantly upregulated in the peripheral blood cells of RCC patients. Importantly, aberrant CXCL7 expression was confirmed even in peripheral blood cells obtained from early stage (pT1a) RCC patients, and the expression level of CXCL7 in peripheral blood cells was a potential independent biomarker for the diagnosis of RCC by receiver operating characteristic curve analysis (sensitivity, 70.0%; specificity, 64.0%; area under the curve = 0.722; multiple logistic regression analysis: odds ratio, 1.07; 95% confidence interval, 1.03-1.11; P = 0.0004). Moreover, CXCL7 expression in peripheral blood cells significantly decreased after resection of the primary tumor. CXCL7 is more highly expressed in PBMCs than in neutrophils from both healthy controls and RCC patients. Interestingly, CXCL7 expression in PBMCs from healthy volunteers was significantly elevated following coculture with RCC cells compared to those cocultured with normal cells as a control. These results suggest that aberrant CXCL7 expression in peripheral blood cells is induced by RCC cells and may serve as a novel biomarker in the diagnosis of RCC.

Anthocyanin Cyanidin-3-Glucoside Attenuates Platelet Granule Release in Mice Fed High-Fat Diets.

Platelet granule release is considered an important target for preventing and treating cardiovascular diseases (CVDs). Cyanidin-3-glucoside (Cy-3-g) is a predominant bioactive anthocyanin compound in many edible plants and has been reported to be protective against CVDs by attenuating platelet dysfunction. However, direct evidence of the action of Cy-3-g on platelet granule secretion in purified platelets from in vivo assays is still poor. In the present study, we demonstrated that dietary supplementation of purified Cy-3-g reduces serum lipid levels and facilitates down-regulation of the platelet granule release of substances such as P-selectin, CD40L, 5-HT, RANTES and TGF-ß1 in gel-filtered platelets, in addition to attenuating serum PF4 and ß-TG levels in mice fed high-fat diets. These results provide evidence that Cy-3-g protects against thrombosis and CVDs by inhibiting purified platelet granule release in vivo.

ß-Thromboglobulin and incident cardiovascular disease risk: The Atherosclerosis Risk in Communities study.

INTRODUCTION: Although it has been suggested that increased concentrations of activated platelet biomarkers are associated with increased risk of incident cardiovascular disease (CVD) in the general population, evidence for this association is still controversial. Thus, we tested the hypothesis that activated platelets, measured by higher concentrations of ß-thromboglobulin, are associated with increased risk of incident CVD (coronary heart disease, heart failure ischemic stroke, and atrial fibrillation). MATERIALS AND METHODS: We prospectively followed a cohort random sample of the Atherosclerosis Risk in Communities (ARIC) cohort, aged 45-64years, and free of CVD at baseline who had previous measurements of plasma ß-thromboglobulin. We identified incident CVD from 1987 through 2013, and used a weighted Cox proportional hazard models to estimate hazard ratios (HRs) and their 95% confidence intervals (CIs). RESULTS: During the 14,387person-years of follow-up for the 746 participants, we identified 140 coronary heart diseases, 123 heart failures, 54 ischemic strokes, and 126 atrial fibrillations. The age-, sex-, and race-adjusted model showed no association between plasma ß-thromboglobulin and CVD, regardless of subtypes. After further adjustment for other CVD risk factors, including antiplatelet agent use, ß-thromboglobulin remained unassociated with CVD risk. CONCLUSIONS: In the prospective population-based ARIC cohort, ß-thromboglobulin was not associated with CVD risk. Our results do not support the hypothesis that a blood marker of higher platelet activity reflects increased future risk of CVD in the general population.

Effect of sublingual immunotherapy on platelet activity in children with allergic rhinitis / Efeito da imunoterapia sublingual sobre a atividade plaquetária em crianças com rinite alérgica

Abstract Introduction: The role of platelet activation in allergic inflammation is receiving increasing attention. Sublingual immunotherapy for allergic rhinitis can modify the immunological process to an allergen, rather than simply treating symptoms. Objective: The aim of this study was to explore the role of platelet activation during sublingual immunotherapy in children with allergic rhinitis. Methods: Forty-two House Dust Mite - sensitized children with allergic rhinitis were enrolled and received House Dust Mite allergen extract for sublingual immunotherapy or placebo. Serum of different time points during treatment was collected and used for detection of Platelet Factor-4 and Beta-Thromboglobulin concentration by Enzyme-Linked Immuno Sorbent Assay. Results: Our data showed decreased expression of Platelet Factor-4 and Beta-Thromboglobulin protein after one year's sublingual immunotherapy. In addition, the decrease of symptom scores and serum Platelet Factor-4 and Beta-Thromboglobulin protein concentrations was positively related. Conclusion: During sublingual immunotherapy, platelet activation was inhibited significantly. Our results might indicate that inhibition of platelet activation within the systemic circulation is an important mechanism during sublingual immunotherapy.

Resumo Introdução: O papel da ativação de plaquetas na inflamação alérgica recebeu atenção crescente. A imunoterapia sublingual para rinite alérgica pode modificar o processo imunológico a um alérgeno, em vez de tratar os sintomas simplesmente. Objetivo: Explorar o papel da ativação plaquetária durante a imunoterapia sublingual em crianças com rinite alérgica. Método: Quarenta e duas crianças com rinite alérgica sensibilizadas por ácaros de poeira domiciliar (APD) foram inscritas e receberam extrato de alérgeno de APD para imunoterapia sublingual ou placebo. O soro de diferentes pontos no tempo durante o tratamento foi recolhido e usado para a detecção de fator 4 plaquetário e concentração de beta-tromboglobulina por ensaio imunoenzimático. Resultados: Nossos dados mostraram diminuição da expressão de fator 4 plaquetário e proteína beta-tromboglobulina após imunoterapia sublingual de um ano. Além disso, a diminuição dos escores de sintomas e o fator 4 plaquetário sérico e concentrações de proteína beta-tromboglobulina foram relacionados de maneira positiva. Conclusão: Durante imunoterapia sublingual, a ativação plaquetária foi inibida significativamente. Os nossos resultados podem indicar que a inibição da ativação de plaquetas dentro da circulação sistêmica é um mecanismo importante durante imunoterapia sublingual.

Effect of sublingual immunotherapy on platelet activity in children with allergic rhinitis.

INTRODUCTION: The role of platelet activation in allergic inflammation is receiving increasing attention. Sublingual immunotherapy for allergic rhinitis can modify the immunological process to an allergen, rather than simply treating symptoms. OBJECTIVE: The aim of this study was to explore the role of platelet activation during sublingual immunotherapy in children with allergic rhinitis. METHODS: Forty-two House Dust Mite - sensitized children with allergic rhinitis were enrolled and received House Dust Mite allergen extract for sublingual immunotherapy or placebo. Serum of different time points during treatment was collected and used for detection of Platelet Factor-4 and Beta-Thromboglobulin concentration by Enzyme-Linked Immuno Sorbent Assay. RESULTS: Our data showed decreased expression of Platelet Factor-4 and Beta-Thromboglobulin protein after one year's sublingual immunotherapy. In addition, the decrease of symptom scores and serum Platelet Factor-4 and Beta-Thromboglobulin protein concentrations was positively related. CONCLUSION: During sublingual immunotherapy, platelet activation was inhibited significantly. Our results might indicate that inhibition of platelet activation within the systemic circulation is an important mechanism during sublingual immunotherapy.

[Optimization of hypolipidemic therapy in patients with ischemic stroke and diabetes mellitus].

OBJECTIVES: Evaluation of effectiveness of Mexidol in optimization of hypolipidemic therapy in ischemic stroke and diabetes mellitus patients. MATERIAL AND METHODS: Authors analyzed the indicators of lipid status: total cholesterol, low-density lipoproteins, high density lipoproteins, triglycerides and concentration of platelet factor-4, ß-tromboglobulin, von Willebrand factor in 68 patients with acute ischemic stroke and diabetes mellitus. Authors investigate the dynamics of these parameters (1(st), 21(st), 3-d and 6(th) month after onset stroke) depending on timing and dose of Mexidol. RESULTS: Long time therapy of Mexidol may optimize of hypolipidemic therapy in ischemic stroke and diabetes mellitus patients.

Differences in intracellular calcium dynamics cause differences in α-granule secretion and phosphatidylserine expression in platelets adhering on glass and TiO2.

In this study, the activation of purified human platelets due to their adhesion on glass and TiO2 in the absence of extracellular calcium was investigated. Differences in α-granule secretion between platelets adhering on the two surfaces were detected by examining the expression and secretion of the α-granule markers P-selectin (CD62P) and ß-thromboglobulin. Similarly, differences in the expression of phosphatidylserine (PS), and in the activation of the major integrin GPIIb/IIIa, on the surfaces of the adhering platelets, were also observed. While all of these activation markers were expressed in platelets adhering on glass, the surface markers were not expressed in platelets adhering on TiO2, and ß-thromboglobulin secretion levels were substantially reduced. Differences in marker expression and secretion correlated with differences in the intracellular calcium dynamics. Calcium ionophore treatment triggered α-granule secretion and PS expression in TiO2-adhering platelets but had no effect on the activation of GPIIb/IIIa. These results demonstrate specificity in the way surfaces of artificial materials activate platelets, link differences in the intracellular calcium dynamics observed in the platelets adhering on the two surfaces to the differences in some of the platelet responses (α-granule secretion and PS expression), but also highlight the involvement of synergistic, calcium-independent pathways in platelet activation. The ability to control activation in surface-adhering platelets makes this an attractive model system for studying platelet signaling pathways and for tissue engineering applications.

Prognostic Significance of Biomarkers in Pulmonary Arterial Hypertension.

RATIONALE: Pulmonary arterial hypertension (PAH) is a rare progressive disease of the pulmonary vasculature that is characterized by endothelial dysfunction, inflammation, and right ventricular dysfunction. OBJECTIVES: The main objective was to determine whether endothelial, inflammatory, and cardiac biomarkers would be associated with the World Health Organization functional assessment and survival in patients with PAH. METHODS: We performed a retrospective cohort study of patients with PAH enrolled in the Randomized Clinical Trial of Aspirin and Simvastatin for Pulmonary Arterial Hypertension (ASA-STAT). Biomarkers (N-terminal fragment of pro-BNP [NT-pro-BNP], von Willebrand factor [vWF], soluble P selectin, C-reactive protein, total and high-density lipoprotein cholesterol, triglycerides, tumor necrosis factor, IL-6, ß-thromboglobulin, and thromboxane B2) were measured at baseline. Patients from the study were followed until lung transplantation, death, or August 1, 2013. Ordinal logistic regression and Cox regression analyses were performed. MEASUREMENTS AND MAIN RESULTS: Sixty-five patients with PAH were enrolled. The mean age was 51 years, and 86% were women. Higher vWF activity, lower high-density lipoprotein cholesterol, and higher thromboxane B2 levels were associated with worse World Health Organization functional class after adjustment for age, sex, and etiology of PAH. Higher NT-pro-BNP levels, lower vWF activity, and lower total cholesterol were associated with an increased risk of death or lung transplant after adjustment for age, sex, etiology of PAH, and 6-minute-walk distance. CONCLUSIONS: In patients with PAH, lower vWF activity and cholesterol levels and higher NT-pro-BNP levels at baseline were associated with an increased risk of death or transplantation. Clinical trial registered with www.clinicaltrials.gov (NCT00384865).

Elevated Release of Beta-thromboglobulin and Platelet Factor 4 in Cerebral Infarction Patients with Branch Atheromatous Disease: A Preliminary Report.

This study evaluated the levels of the platelet activation markers beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) in patients with branch atheromatous disease (BAD). Patients with newly diagnosed cerebral infarctions were recruited into the study; those with cardiogenic cerebral infarctions were excluded. Beta-TG and PF4 levels were measured before therapeutic intervention and compared between patients with and without BAD; Welch's t-test was used to determine significant differences between the groups. A total of 15 subjects were enrolled in the study, and 8 were diagnosed with BAD. Beta-TG (P = 0.031) and PF4 (P = 0.041) levels were significantly higher in the BAD patients than in the non-BAD patients. Platelet activity is normally elevated in patients with cerebral infarctions, but is elevated to an even greater extent in BAD patients. The evaluation of beta-TG and PF4 levels may be beneficial for the elucidation of BAD.

Elevated expression of platelet-derived chemokines in patients with antiphospholipid syndrome.

OBJECTIVE: Platelet factor 4 tetramers (CXCL4 chemokine) form complexes with ß2glycoprotein I (ß2GPI), recognized by anti-ß2GPI antibodies leading to platelet activation in antiphospholipid syndrome (APS), either primary (PAPS) or secondary (SAPS). Increased plasma levels of CXCL4 may favor this process; therefore we measured plasma levels of CXCL4, a CXCL4 variant (CXCL4L1) and as controls, platelet-derived chemokines CXCL7 (NAP-2) and CCL5 (RANTES), in APS, and disease controls such as patients with systemic lupus erythematosus (SLE) coronary artery disease (CAD) and healthy donors (HDs). METHODS: Plasma samples and platelets were isolated from patients with APS (n = 87), SLE (n = 29), CAD (n = 14) and 54 HDs. Plasma levels of CXCL4, CXCL4L1, CXCL7 and CCL5 as well as intracellular platelet CXCL4 and CXCL4L1 were measured using ELISA. Platelet CXCL4 and CXCL4L1 RNA levels were determined by RT-PCR. RESULTS: CXCL4, CXCL7 (NAP-2) and CCL5 (RANTES) plasma levels were significantly higher in patients with APS compared to both control groups (SLE, CAD) and HDs. CXCL4L1 plasma levels were also significantly higher in APS than in SLE and HDs, but lower from that of CAD patients. Statistically significant concordance was detected between CXCL4 and CXCL7 (p < 0.0001) or CCL5 (p < 0.0001) plasma levels in patients with APS, either PAPS or SAPS. CXCL4L1 plasma levels were inversely correlated with CXCL4 (P = 0.0027), CXCL7 (p = 0.012) and CCL5 (p = 0.023) in PAPS and positively with CXCL4 (p = 0.0191), CCL5 (p < 0.0001) and CXCL7 (P < 0.0001), in SAPS. Levels of CXCL4, CXCL4L1, CXCL7 and CCL5 were divided in "high" (exceeding a level defined as the mean of HDs and 3 SD) and "low" (below this level); The "CXCL4L1 high" group was characterized by increased IgG aCL, (p = 0.0215), double antibody positivity (either aCL or anti-ß2GPI plus LA), (p = 0.0277), triple antibody positivity (aCL plus anti-ß2GPI plus LA), (p = 0.0073) and thrombocytopenia (p = 0.0061), as well as with at least 1 thrombotic event or the last 5 years (p = 0.0001), or more than 3 thrombotic events ever (p = 0.0151). CONCLUSIONS: Chemokines associated with platelet activation and immune cell chemotaxis were found to be elevated in APS patients' plasma and may contribute to the pathogenesis of the syndrome. High CXCL4L1 plasma levels are associated with the clinical expression of APS and should be prospectively evaluated as a biomarker.

Novel TIA biomarkers identified by mass spectrometry-based proteomics.

BACKGROUND: Transient ischemic attacks remain a clinical diagnosis with significant variability between physicians. Finding reliable biomarkers to identify transient ischemic attacks would improve patient care and optimize treatment. AIM: Our aim is to identify novel serum TIA biomarkers through the use of mass spectroscopy-based proteomics. METHODS: Patients with transient neurologic symptoms were prospectively enrolled. Mass spectrometry-based proteomics, an unbiased method to identify candidate proteins, was used to test the serum of the patients for biomarkers of cerebral ischemia. Three candidate proteins were found, and serum concentrations of these proteins were measured by enzyme-linked immunosorbent assay in a second cohort of prospectively enrolled patients. The Student's t-test was used for comparison. The Benjamini-Hochberg false discovery rate controlling procedure for multiple comparison adjustments determined significance for the proteomic screen. RESULTS: Patients with transient ischemic attacks (n = 20), minor strokes (n = 15), and controls (i.e. migraine, seizure, n = 12) were enrolled in the first cohort. Ceruloplasmin, complement component C8 gamma (C8γ), and platelet basic protein were significantly different between the ischemic group (transient ischemic attack and minor stroke) and the controls (P = 0·0001, P = 0·00027, P = 0·00105, respectively). A second cohort of patients with transient ischemic attack (n = 22), minor stroke (n = 20), and controls' (n = 12) serum was enrolled. Platelet basic protein serum concentrations were increased in the ischemic samples compared with control (for transient ischemic attack alone, P = 0·019, for the ischemic group, P = 0·046). Ceruloplasmin trended towards increased concentrations in the ischemic group (P = 0·127); no significant difference in C8γ (P = 0·44) was found. CONCLUSIONS: Utilizing mass spectrometry-based proteomics, platelet basic protein has been identified as a candidate serum biomarker for transient ischemic attack. This unbiased proteomic approach may be a promising method to identify novel biomarkers to more precisely diagnose transient ischemic attacks.

Platelet Activation Markers Are Associated with Crohn's Disease Activity in Patients with Low C-Reactive Protein.

BACKGROUND: In assessing Crohn's disease (CD) activity, C-reactive protein (CRP) is an important indicator of inflammation; however, it is not necessarily associated with the Crohn's Disease Activity Index (CDAI), particularly in patients with low CRP. Recently, platelet activation factors have been recognized due to their importance in the inflammatory response. In this study, we examined associations between the CDAI and platelet factor 4 (PF-4), ß-thromboglobulin (ß-TG), and other coagulation and fibrinolysis factors. AIMS: We aimed to find a new marker for evaluating disease activity in patients with CD and low CRP. METHODS: Nine markers, including CRP, platelet count, white blood cell count, fibrin and fibrinogen degradation product, fibrinogen, thrombin-antithrombin complex, prothrombin fragments 1 + 2, PF-4, and ß-TG were evaluated in 47 patients with CD and low CRP (<1.0 mg/dl). Patients were assigned to high or low disease activity groups, CDAI-H (CDAI ≥ 150) and CDAI-L (CDAI < 150), respectively. RESULTS: CDAI-H exhibited significantly higher PF-4 and ß-TG levels than CDAI-L (P < 0.01). Other markers were not significantly different between groups. CDAI was positively correlated with the levels of PF-4 and ß-TG (P = 0.0033 and 0.0024; r = 0.4202 and 0.4321, respectively). Receiver operating characteristic curve analyses of PF-4 and ß-TG showed high sensitivity (61.9 and 81%, respectively) and specificity (84.7 and 69.2%, respectively) for diagnosing active CD. CONCLUSION: Among eight potential markers, PF-4 and ß-TG were the most highly correlated with CDAI in patients with CD and low CRP. PF-4 and ß-TG levels showed promise as new markers for assessing CD in patients with low CRP.

Circulating platelet-neutrophil aggregates play a significant role in Kawasaki disease.

BACKGROUND: Circulating platelet-neutrophil aggregates play a crucial role in amplifying acute inflammation and could promote adverse effects involving vascular injury. The aim of this study was to evaluate the role of platelet-neutrophil aggregates in Kawasaki disease (KD). METHODS AND RESULTS: Forty patients with KD (30 intravenous immunoglobulin [IVIG] responders and 10 IVIG non-responders), 7 febrile patients with bacterial infections, and 9 normal volunteers were analyzed. Thirty-three patients with KD were treated with IVIG, and 7 were treated with IVIG plus prednisolone. We evaluated the rate of platelet-neutrophil aggregates and measured the platelet factor 4 (PF4) and ß-thromboglobulin (ß-TG) levels. The rate of platelet-neutrophil aggregates was significantly higher in patients with KD than those with bacterial infection and normal volunteers. The rate of platelet-neutrophil aggregates was significantly higher in patients with coronary artery abnormalities (CAA) than in those without CAA, and was correlated with PF4 and ß-TG levels in patients with KD. Comparing time-course analysis, the rate of platelet-neutrophil aggregates was significantly decreased in patients treated with IVIG plus prednisolone than in those treated with IVIG alone. CONCLUSIONS: The findings demonstrate that platelet-neutrophil aggregates are significantly present in higher rates and are closely related to pathological developments of CAA in KD. Additional prednisolone treatment for patients in the acute phase of KD could suppress platelet-neutrophil aggregates, indicating that platelet-neutrophil aggregates would inhibit amplified reciprocal vascular inflammatory activation.