Cellular mechanisms involved in the pathogenesis of airway remodeling in chronic lung disease.

Background: Airway epithelial cells and lung fibroblasts play an important role in the development of chronic lung disease, but the exact mechanisms responsible have not been clarified. Our objective was to investigate the involvement of these cells in the inflammatory response associated to chronic lung disease. Methods: Human lung fibroblasts and airway epithelial cells were challenged with Interleukin-1ß and hypoxia, and with inhibitory (simvastatin) stimuli of the inflammatory response. Expression of markers of local inflammation ((IL-8, monocyte chemoattractant protein-1 (MCP-1), factor-κB1 (NF-κB1)), systemic inflammation ((C-reactive protein (CRP) and serum amyloid A (SAA)) and proteases matrix metalloproteinase (MMP) 9 and 12 were assessed by PCR and ELISA. Apoptosis/necrosis was analyzed by flow cytometry. Results: Our results showed that the lung fibroblasts had a higher expression of local and systemic inflammation and protease activity markers when they were treated with IL-1ß compared to airway epithelial cells. Under hypoxic conditions, we observed a decrease in systemic inflammation in lung fibroblasts, which was further attenuated by simvastatin. Conclusion: The lung fibroblasts seem to be the main initially stimulated cells that could potentially trigger the inflammatory response, and be responsible for the eventual onset of chronic lung disease. The involvement of IL-1ß stimulation in systemic inflammatory and proteinase imbalance biomarkers is higher in lung fibroblasts. Apoptosis is not a predominant mechanism in these cells.

Differential biomarker profiles between unprovoked venous thromboembolism and cancer.

BACKGROUND: The relationship between cancer and venous thromboembolic disease (VTD) are complex because the activated coagulation factors are not only involved in thrombosis but also in malignant processes, such as angiogenesis and metastasis. OBJECTIVE: To compare phenotypes of extracellular vesicles (EVs), and levels of D-dimer, soluble P-selectin (sP-selectin) and antigenic tissue factor (TF) between unprovoked VTD patients, who did not develop cancer during one-year follow-up, and those with advanced stage of cancer but not associated with VTD. METHODS: A prospective study in which we included 138 unprovoked VTD patients and 67 advanced cancer patients, who did not develop thrombosis. Levels of EVs of different cellular origin (platelet, endothelium and leukocyte), EVs positive for tissue factor (TF) and P-selectin glycoprotein ligand 1 were quantified by flow cytometry. D-dimer, soluble P-selectin (sP-selectin) and antigenic TF were determined by ELISA. RESULTS: TF-positive EVs, D-dimer, and sP-selectin were markedly elevated in unprovoked VTD patients compared to cancer patients without association with thrombosis. CONCLUSIONS: Levels of TF-positive EVs, D-dimer and sP-selectin are able to discriminate between unprovoked VTD patients not related to cancer and cancer patients not associated with VTD. These results could lead to the application of EVs as biomarkers of both diseases. Key messages: Circulating EVs, specifically TF-positive EVs, in combination with plasmatic markers of hypercoagulable states, such as D-dimer, sP-selectin and antigen TF, are able to discriminate between cancer patients without thrombosis and patients with unprovoked VTD. Research fields could be opened. Future studies will assess if these biomarkers together serve as predicting thrombotic events in cancer populations.

Evaluation of liposome populations using a sucrose density gradient centrifugation approach coupled to a continuous flow system.

A method for the evaluation of liposome size populations using sucrose density gradient centrifugation coupled with a continuous flow system is presented. Liposomes, prepared using different methods (rapid solvent evaporation, rehydration, and detergent removal) and modified by assaying several procedures (shaking, sonication and extrusion) were evaluated according to the type of liposome, size and polydispersity. The preparation of liposomes was carried out in the presence of the fluorophor cresyl violet. Extracts of the liposomes were homogenised and centrifuged at 20,073 x g at 4 degrees C for 30 min using sucrose density gradient centrifugation programmes, which provide efficient liposome separation in different sizes. The results of the separation procedure were tested by aspiration of the extracts into a continuous flow system in which the liposomes were disrupted by the continuous mixing with a Triton X-100 solution, prior to their translation to the detector. The luminescence provided by the liberation of the encapsulated fluorophor indicates the distribution of liposomes in each density gradient stage. Three zones were obtained: zone alpha, containing giant unilamellar and multivesicular vesicles, zone beta, with large and medium size liposomes, and zone gamma, which contained small size liposomes. The precision of the separation zones obtained, expressed as RSD%, was lower than 5.6% in all instances. The method provides a relative rapid way to evaluate the liposome polydispersity and size after using conventional methods of synthesis and mechanical modifications.