Verónica Sánchez-López

C-reactive protein and serum amyloid a overexpression in lung tissues of chronic obstructive pulmonary disease patients: a case-control study.

Background. Although researchers have consistently demonstrated systemic inflammation in chronic obstructive pulmonary disease (COPD), its origin is yet unknown. We aimed to compare the lung bronchial and parenchymal tissues as potential sources of major acute-phase reactants in COPD patients and resistant smokers. Methods. Consecutive patients undergoing elective surgery for suspected primary lung cancer were considered for the study. Patients were categorized as COPD or resistant smokers according to their spirometric results. Lung parenchyma and bronchus sections distant from the primary lesion were obtained. C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2 and SAA4) gene expressions were evaluated by RT-PCR. Protein levels were evaluated in paraffin embedded lung tissues by immunohistochemistry and in serum samples by nephelometry. Results. Our study included 85 patients with COPD and 87 resistant smokers. In bronchial and parenchymal tissues, both CRP and SAA were overexpressed in COPD patients. In the bronchus, CRP, SAA1, SAA2, and SA4 gene expressions in COPD patients were 1.89-fold, 4.36-fold, 3.65-fold, and 3.9-fold the control values, respectively. In the parenchyma, CRP, SAA1, and SAA2 gene expressions were 2.41-, 1.97-, and 1.76-fold the control values, respectively. Immunohistochemistry showed an over-stained pattern of these markers on endovascular cells of COPD patients. There was no correlation with serum protein concentration. Conclusions. These results indicate an overexpression of CRP and SAA in both bronchial and parenchymal tissue in COPD, which differs between both locations, indicating tissue/cell type specificity. The endothelial cells might play a role in the production of theses markers.

Differential expression of C-Reactive protein and Serum amyloid A in different cell types in the lung tissue of chronic obstructive pulmonary disease patients

BackgroundChronic systemic inflammatory syndrome has been implicated in the pathobiology of extrapulmonary manifestations of chronic obstructive pulmonary disease (COPD). We aimed to investigate which cell types within lung tissue are responsible for expressing major acute-phase reactants in COPD patients and disease-free (“resistant”) smokers.MethodsAn observational case–control study was performed to investigate three different cell types in surgical lung samples of COPD patients and resistant smokers via expression of the C-reactive protein (CRP) and serum amyloid A (SAA1, SAA2 and SAA4) genes. Epithelial cells, macrophages and fibroblasts from the lung parenchyma were separated by magnetic microbeads (CD326, CD14 and anti-fibroblast), and gene expression was evaluated by RT-PCR.ResultsThe sample consisted of 74 subjects, including 40 COPD patients and 34 smokers without disease. All three cell types were capable of synthesizing these biomarkers to some extent. In fibroblasts, gene expression analysis of the studied biomarkers demonstrated increased SAA2 and decreased SAA1 in patients with COPD. In epithelial cells, there was a marked increase in CRP, which was not observed in fibroblasts or macrophages. In macrophages, however, gene expression of these markers was decreased in COPD patients compared to controls.ConclusionsThese results provide novel information regarding the gene expression of CRP and SAA in different cell types in the lung parenchyma. This study revealed differences in the expression of these markers according to cell type and disease status and contributes to the identification of cell types that are responsible for the secretion of these molecules.

Cigarette Smoke Decreases the Maturation of Lung Myeloid Dendritic Cells.

Background Conflicting data exist on the role of pulmonary dendritic cells (DCs) and their maturation in patients with chronic obstructive pulmonary disease (COPD). Herein, we investigated whether disease severity and smoking status could affect the distribution and maturation of DCs in lung tissues of patients undergoing elective pneumectomy or lobectomy for suspected primary lung cancer. Materials and Methods A total of 75 consecutive patients were included. Spirometry testing was used to identify COPD. Lung parenchyma sections anatomically distant from the primary lesion were examined. We used flow cytometry to identify different DCs subtypes—including BDCA1-positive myeloid DCs (mDCs), BDCA3-positive mDCs, and plasmacytoid DCs (pDCs)—and determine their maturation markers (CD40, CD80, CD83, and CD86) in all participants. We also identified follicular DCs (fDCs), Langerhans DCs (LDCs), and pDCs in 42 patients by immunohistochemistry. Results COPD was diagnosed in 43 patients (16 current smokers and 27 former smokers), whereas the remaining 32 subjects were classified as non-COPD (11 current smokers, 13 former smokers, and 8 never smokers). The number and maturation of DCs did not differ significantly between COPD and non-COPD patients. However, the results of flow cytometry indicated that maturation markers CD40 and CD83 of BDCA1-positive mDCs were significantly decreased in smokers than in non-smokers (P = 0.023 and 0.013, respectively). Immunohistochemistry also revealed a lower number of LDCs in COPD patients than in non-COPD subjects. Conclusions Cigarette smoke, rather than airflow limitation, is the main determinant of impaired DCs maturation in the lung.

Stent‐induced tracheal stenosis can be predicted by IL‐8 expression in rabbits

Bare metal stents may cause complications like fibrous encapsulation, granulation and tracheal stenosis. We investigated the behaviour of three commercially available stents in vivo (rabbits) and in vitro (coculture of those stents with epithelial and fibroblast cell lines). Also, we investigated whether development of tracheal stenosis could be predicted by any biological marker.

Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters.

Abstract Background: Microparticles (MPs) have been shown to be markers of cellular activation and interactions. Pre-analytical conditions such as the centrifugation protocol and sample storage conditions represent an important source of variability in determining MPs values. The objectives of this study were to evaluate the influence of sample storage conditions and centrifugation speed and temperature on the determination of MPs in plasma. Methods: Citrate-anticoagulated blood samples obtained from 21 healthy subjects were centrifuged under four different protocols involving different speeds (2500 g or 1500 g) and temperatures (4 °C or 20 °C) to isolate platelet-poor plasma (PPP). The number of MPs in fresh and frozen-thawed PPP were analyzed by flow cytometry, and MPs-mediated procoagulant activity was determined by a thrombin generation test and phospholipid-dependent procoagulant tests. Results: The number of MPs and their procoagulant activity were affected by freeze-thaw cycling and centrifugation speed but not by centrifugation temperature. Sample freezing increased MPs number (six-fold) and thrombin generation (four-fold), and decreased clotting time (two-fold). Low centrifugation speed caused an increase in MPs number and a parallel increase in MP-mediated procoagulant activity. Conclusions: Sample storage conditions and centrifugation speed are important processing conditions affecting MPs number and activity. Before any study, the protocol for MPs isolation should be optimized to ensure a reliable characterization of MPs, which could provide important information for diagnostic purposes and for understanding the pathogenesis of diseases.

High correlation between 2 flow cytometry platforms in the microparticles analysis using a new calibrated beads strategy

Microparticles (MPs) are potential noninvasive biomarkers for diagnosis or prognosis in pathologic conditions. However, the lack of standardization of the preanalytical and analytical methods leads to a wide variability in MPs results. The recently developed Megamix-Plus beads, a new bead-based standardization tool optimized to specific types of flow cytometers, could help circumvent this problem. The aim of the present study was to determine whether the number of total MPs and platelet-derived MPs (PMPs) is similar using 2 different cytometer platforms calibrated with the Megamix-Plus beads. Blood samples from 65 patients with deep venous thrombosis were collected and processed to obtain platelet poor plasma (PPP). The number of total MPs and PMPs in each PPP sample was measured using 2 flow cytometers. Megamix-Plus side scatter channel beads were used to calibrate the LSRFortessa flow cytometer from Becton Dickinson, whereas Megamix-Plus forward scatter channel beads were applied to the Navios flow cytometer from Beckman Coulter. High correlation of total MPs and PMPs values between the flow cytometers was found (r = 0.908, P < 0.01 and r = 0.910, P < 0.001, respectively). However, the absolute numbers of total MPs and PMPs were significantly higher measured with the Navios flow cytometer compared with the LSRFortessa cytometer. Therefore, both platforms are valid for MPs determination in general, although a similar platform with the same calibration tool could be a better choice for multicenter studies.

Survivin is a negative prognostic factor in malignant pleural effusion

Survivin is a well‐known member of the inhibitor of apoptosis family, and has been related to increased tumour aggressivity, both in tissue and in pleural fluid.

D-dimer and high-sensitivity C-reactive protein levels to predict venous thromboembolism recurrence after discontinuation of anticoagulation for cancer-associated thrombosis

BackgroundOptimal duration of anticoagulation for cancer-associated thrombosis (CAT) remains unclear. This study assessed D-dimer (DD) and high-sensitivity C-reactive protein (hs-CRP) levels after the withdrawal of anticoagulation treatment to predict the risk of venous thromboembolism (VTE) recurrence among patients with CAT.MethodsProspective, multicentre study to evaluate CAT with ≥3 months of anticoagulation that was subsequently discontinued. Blood samples were taken when patients stopped the anticoagulation and 21 days later to determine the DD and hs-CRP levels. All patients were followed up for 6 months to detect VTE recurrence.ResultsBetween 2013 and 2015, 325 patients were evaluated and 114 patients were ultimately enrolled in the study. The mean age was 62 ± 14 years and nearly 40% had metastasis. Ten patients developed VTE recurrence within 6 months (8.8%, 95% confidence interval [CI]: 4.3–15.5%). The DD and hs-CRP levels after 21 days were associated with VTE recurrence. The subdistribution hazard ratios were 9.82 for hs-CRP (95% CI: 19–52) and 5.81 for DD (95% CI: 1.1–31.7).ConclusionsThis study identified that hs-CRP and DD were potential biomarkers of VTE recurrence after discontinuation of anticoagulation in CAT. A risk-adapted strategy could identify low-risk patients who may benefit from discontinuation of anticoagulation.

Specific networks of plasma acute phase reactants are associated with the severity of chronic obstructive pulmonary disease: a case-control study

Objectives. A detailed understanding of the intricate relationships between different acute phase reactants (APRs) in chronic obstructive pulmonary disease (COPD) can shed new light on its clinical course. In this case-control study, we sought to identify the interaction networks of a number of plasma APRs in COPD, with a special focus on their association with disease severity. Methods. COPD cases and healthy smoking controls (3:1 ratio) were recruited in our outpatient pulmonary clinic. Cardiopulmonary exercise testing was used to rule out the presence of ischemic heart disease. All subjects were males as per protocol. Multiple plasma APRs - including α-2-macroglobulin, C-reactive protein (CRP), ferritin, fibrinogen, haptoglobin, procalcitonin (PCT), serum amyloid A (SAA), serum amyloid P, and tissue plasminogen activator (tPA) - were measured using commercial Acute Phase Bio-Plex Pro Assays and analyzed on the Bio-Plex manager software. Correlations between different APRs were investigated using a heat map. Network visualization and analyses were performed with the Cytoscape software platform. Results. A total of 96 COPD cases and 33 controls were included in the study. Plasma A2M, CRP, and SAP levels were higher in COPD patients than in controls. Circulating concentrations of haptoglobin and tPA were found to increase in parallel with the severity of the disease. Increasing disease severity was associated with distinct intricate networks of APRs, which were especially evident in advanced stages. Conclusions. We identified different networks of APRs in COPD, which were significantly associated with disease severity.

C0280 Correlation between annexinV-FITC and Annexin V-V450 positive microparticles in two high sensitivity cytometers

Background: Thromboelastometry (TEM) has the advantage of studying whole blood (WB) or platelet rich plasma (PRP), in contrast to thrombin generation test (TGT) which cannot be performed in WB. The aim is to investigate the response in ROTEM® delta and TGT to enoxaparin, dabigatran and rivaroxaban and determine if these different methods show a dose/response relationship. A correlation between ROTEM (WB or PRP) and TGT parameters is also investigated. Methods: Citrated WB was obtained from 18 healthy volunteers from a blood bank and tested in ROTEM with increasing concentrations between 0 and 0.5 μg/mL for dabigatran and rivaroxaban, and 0 and 0.5 anti-Xa IU/mL for enoxaparin. After spinning theWB, the PRP obtained (without adjusting platelet count) has been tested in ROTEM and TGT with the 3 anticoagulants studied. TEM and TGT were triggered with tissue factor: 0.36 and 1 pM final respectively. Statistical analysis has been performed with XLSTAT software. Results: Parameters which show the best correlation to the anticoagulant doses are CT for ROTEM and LT for TGT with a Spearman rhoN0.9. The 3 drugs influence the other parameters in a weak dose-dependent manner, except for ROTEM amplitude and TGT ETP which are not modified. In general, CT in WB is more sensitive than in PRP. The concentrations needed to prolong the ROTEM CT 2 fold (Tx2) are 0.27 IU/mL, 0.06 and 0.20 μg/mL inWBversus 0.38 IU/mL, 0.10 and 0.18 μg/mL in PRP for enoxaparin, dabigatran and rivaroxaban respectively. TGT LT is less sensitive with Tx2 at 0.50 IU/mL, 0.15 and 0.30 μg/mL for enoxaparin, dabigatran and rivaroxaban respectively. The statistical analysis show a good correlation between WB and PRP in ROTEM CT, and between ROTEM CT and TGT LT with a Spearman rhoN0.9. Comment: TGT and ROTEM are influenced by enoxaparin, dabigatran and rivaroxaban concentrations in a significant dose/response relationship for time parameters. The differentmethods studied could be used for the evaluation of the anticoagulation level. ROTEM CT was found more sensitive than TGT LT for the 3 anticoagulants. According to these in vitro results, it is likely that both tests may be used as a functional test in treated patients.