Silvia Lepore

Isolation and characterization of microparticles in sputum from cystic fibrosis patients

BackgroundMicroparticles (MPs) are membrane vesicles released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. Cystic fibrosis (CF) lung disease is characterized by massive neutrophil granulocyte influx in the airways, their activation and eventually apoptosis. We investigated on the presence and phenotype of MPs in the sputum, a rich non-invasive source of inflammation biomarkers, of acute and stable CF adult patients.MethodsSpontaneous sputum, obtained from 21 CF patients (10 acute and 11 stable) and 7 patients with primary ciliary dyskinesia (PCD), was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, and identified in the supernatants of treated sputum by cytofluorimetry and immunolabelling for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens.ResultsElectron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria, confirming microbiological tests. CF sputa contained higher number of MPs in comparison with PCD sputa. Levels of CD11a+-and CD66b+-, but not CD11b+-MPs were significantly higher in CF than in PCD, without differences between acute and stable patients.ConclusionsIn summary, MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. This novel isolation method for MPs from sputum opens a new opportunity for the study of lung pathology in CF.

Hematopoietic Stem/Progenitor Cells Express Functional Mitochondrial Energy-Dependent Cystic Fibrosis Transmembrane Conductance Regulator

Bone marrow-derived hematopoietic stem/progenitor cells (HSPCs) encompass a wide array of cell subsets with different capacities of engraftment and injured tissue-regenerating potential. The characterization/isolation of the stem cell subpopulations represents a major challenge to improve the efficacy of transplantation protocols used in regenerative medicine. Cystic fibrosis (CF) is one of the diseases whose hope of cure relies on the successful application of cell-based gene therapy. This study was aimed at characterizing murine HSPCs on the basis of their bioenergetic competence and CF transmembrane conductance regulator (CFTR) expression. Positively immunoselected Sca-1(+) HSPCs encompassed 2 populations distinguished by their different size, Sca-1 expression and mitochondrial content. The smaller were the cells, the higher was Sca-1 expression and the lower was the intracellular density of functional mitochondria. Reverse transcription-polymerase chain reaction and western blotting revealed that HSPCs expressed CFTR mRNA and protein, which was also functional, as assessed by spectrofluorimetric and patch-clamp techniques. Inhibition of mitochondrial oxidative phosphorylation by oligomycin resulted in a 70% decrease of both the intracelluar adenosine triphosphate content and CFTR-mediated channel activity. Finally, HSPCs with lower Sca-1 expression and higher mitochondrial content displayed higher CFTR levels. Our findings identify 2 subpopulations in HSPCs and unveil a so-far unappreciated relationship between bioenergetic metabolism and CFTR in HSPC biology.

Pro-inflammatory effect of cystic fibrosis sputum microparticles in the murine lung

BACKGROUND The role of microparticles (MPs) in the inflammatory process of cystic fibrosis (CF) airways is not known. Here, we have studied the pro-inflammatory potential of CF MPs in a model of acute lung injury. METHODS Swiss mice were subjected to intratracheal administration of MPs obtained from CF and primary ciliary diskinesia (PCD) patients. Histopathology, total and differential cell counts in bronchoalveolar lavage fluid were used to evaluate the inflammatory reaction in the lung. Lipopolysaccharide (LPS)-like activity of MPs was studied by Limulus amebocyte lysate assay. RESULTS MPs obtained from acute CF patients determined peribronchial and perivascular inflammatory infiltrates similar to those elicited by LPS. This inflammation was granulocyte-dominated and higher than that determined by MPs obtained from stable CF, whereas PCD MPs caused a macrophage-dominated inflammation. While LPS-activity was not found in circulating blood MPs prepared from CF patients, it was present in MPs obtained from CF sputum and sputum CD66b(+) neutrophils. Finally, LPS-like activity was only detected in circulating MPs after incubation with LPS as well as in MPs obtained from LPS-stimulated neutrophils obtained from healthy donors. CONCLUSIONS These data suggest that the pro-inflammatory potential of neutrophil-derived MPs in the CF airways may be subsequent to the binding of shedded LPS.

Beta-catenin and epithelial tumors: a study based on 374 oropharyngeal cancers.

Introduction. Although altered regulation of the Wnt pathway via beta-catenin is a frequent event in several human cancers, its potential implications in oral/oropharyngeal squamous cell carcinomas (OSCC/OPSCC) are largely unexplored. Work purpose was to define association between beta-catenin expression and clinical-pathological parameters in 374 OSCCs/OP-SCCs by immunohistochemistry (IHC). Materials and Methods. Association between IHC detected patterns of protein expression and clinical-pathological parameters was assessed by statistical analysis and survival rates by Kaplan-Meier curves. Beta-catenin expression was also investigated in OSCC cell lines by Real-Time PCR. An additional analysis of the DNA content was performed on 22 representative OSCCs/OPSCCs by DNA-image-cytometric analysis. Results and Discussion. All carcinomas exhibited significant alterations of beta-catenin expression (P < 0.05). Beta-catenin protein was mainly detected in the cytoplasm of cancerous cells and only focal nuclear positivity was observed. Higher cytoplasmic expression correlated significantly with poor histological differentiation, advanced stage, and worst patient outcome (P < 0.05). By Real-Time PCR significant increase of beta-catenin mRNA was detected in OSCC cell lines and in 45% of surgical specimens. DNA ploidy study demonstrated high levels of aneuploidy in beta-catenin overexpressing carcinomas. Conclusions. This is the largest study reporting significant association between beta-catenin expression and clinical-pathological factors in patients with OSCCs/OPSCCs.

Evaluation of Genome-Wide Expression Profiles of Blood and Sputum Neutrophils in Cystic Fibrosis Patients Before and After Antibiotic Therapy

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.

Effect of acute lung injury on VLA-4 and CXCR4 expression in resident and circulating hematopoietic stem/progenitor cells

Background: The effect of acute lung injury on adhesion molecule expression in hematopoietic stem/progenitor cells (HSPCs) is poorly understood. Objectives: The aim of this study was to determine whether there is a relationship -between pulmonary inflammation, expression of VLA-4 (CD49d), LFA-1 (CD11a), L-selectin (CD62L), CXCR4, and chemotaxis in resident HSPCs, as well as the level of circulating HSPCs. Methods: Following intratracheal administration of a single LPS bolus in C57Bl/6 mice, the number of inflammatory cells, differential counts, and amounts of cytokines/ chemokines were studied in cytospins and bronchoalveolar lavage fluid (BALF) specimens. Expressions of adhesion -molecules and CXCR4 were analyzed in HSPCs by flow cytometry, as well as SDF-1-directed chemotaxis. Levels of HSPCs in the blood were studied in ungated and circulating subpopulations. Results: In coincidence with a peak of airway neutrophils, cytokine (IL-1β, TNF-α, and IL-6), chemokine (KC, MIP-2, and SDF-1) levels in BALF and the number of marrow HSPCs expressing CD49d and CXCR4 significantly increased at 48 h. The number of CD49d- and CXCR4-positive HSPCs dropped at 72 h. The HSPC subset comprising bigger cells behaved the same for CD49d. Chemotaxis of the marrow HSPC subset of bigger cells was higher in LPS-treated animals than in controls at 72 h. Finally, we could detect a significant decrease in circulating Sca-1+ cells in the mononuclear population at 72 h in LPS-treated mice. Conclusions: Our data provide evidence for a temporal relationship between pulmonary inflammation, CD49d and CXCR4 expression fluctuation in resident HSPCs, and the level of circulating HSPCs.

Stimulation of β2-adrenergic receptor increases CFTR function and decreases ATP levels in murine hematopoietic stem/progenitor cells

BACKGROUND The chloride channel CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) is expressed by many cell types, including hematopoietic stem/progenitor cells (HSPCs). In this study, we sought to better comprehend the regulation of CFTR activity in HSPCs, namely by beta-adrenergic stimuli. METHODS The expression of β2-adrenergic receptor (β2-AR) in murine Sca-1(+) HSPCs was investigated by immunofluorescence/confocal microscopy and flow-cytometric analysis. Association with CFTR was assessed by immunoprecipitation. HSPCs were evaluated for ATP content and CFTR activity by means of luminometric and spectrofluorometric methods, respectively, upon stimulation with salbutamol. RESULTS HSPCs express β2-AR over the whole plasma membrane and are associated in cellula with both the immature and mature forms of CFTR. β2-AR was predominantly expressed by HSPCs with bigger size. CFTR channel activity was increased by salbutamol treatment and this activation was inhibited by either a specific CFTR inhibitor (CFTRinh172) or a β2-AR receptor inhibitor (ICI 118,551). Intracellular ATP levels were reduced by salbutamol stimulation and this effect was reversed when ICI 118,551 or CFTR inhibitors were present. A trend in the increase of extracellular ATP upon salbutamol stimulation was observed. CONCLUSIONS In HSPCs, CFTR is regulated by β2-adrenergic receptor stimulation determining intracellular ATP depletion.

Adhesion and growth of osteoblast-like cells on laserengineered porous titanium surface. Expression and localization of N-cadherin and β-catenin

Response of different types of cells on biomaterials is crucial for the applications of tissue engineering and regenerative medicine. It is recognized that cell behaviour depends largely on material surface characteristics. The purpose of this study was to define the biologic response of MG63 cells to the innovative patented surface SYNTHEGRA. MG63 morphology and distribution on the three different titanium disk surfaces (sandblasted, smooth, and laser-treated) were evaluated by microscopy analysis after staining with hematoxylin and eosin. Cell adhesion was determined by crystal violet assay at 48 h while proliferation and cytotoxicity were performed by MTT assay at 24, 48, 72 and 240 h. The expression and localization of N-cadherin and beta-catenin were studied by immunofluorescence and confocal microscopy. At 48 h the adhesion was similar in all titanium surfaces, no difference in cell viability were observed in all titanium disks when compared with controls, while the cell growth on laser-treated disks was significantly higher at 240 h than at 24 and 72 h. Morphological analysis show that cells are aligned along the grooves and inside the cavities. beta-catenin signal appeared more diffuse and localized underneath the cell membrane, while N-cadherin signal was fainter in cells grown on SYNTHEGRA surface. This work put into evidence the performance of newly designed laser-micromachined surface for adhesion, growth and distribution of human osteoblast-like cells. SYNTHEGRA surface inducing modification of N-cadherin and beta-catenin expression and localization, are suggestive of cells undergoing differentiation towards osteocytes and could be particularly suited for immediate load implant procedures.

Inflammatory effect of sputum microparticles derived from pulmonary diseases in a murine model

Microparticles (MPs) are small plasma membrane vesicles released by several cell types (macrophages, platelets, endothelial cells, granulocytes, monocytes, lymphocytes) following chemical, physical and apoptotic stimuli. MPs bear a number of bioactive effectors that can be disseminate, exchanged, and transferred via MPs cell interactions. The hallmarks of lung disease in cystic fibrosis (CF) patients are a persistent infection with opportunistic bacterial pathogens such as the Gram-negative P. aeruginosa and an abnormal inflammatory response dominated by polymorphonuclear granulocytes (neutrophils). In CF airways, neutrophils undergo conventional activation and functional reprogramming. In our previous study, we isolated MPs in sputum and demonstrated that most of CF sputum MPs were of granulocyte origin. However, the neutrophil response is not capable to clear bacteria from the CF airways ensuing in exaggerated apoptosis. The aim of this study is to investigate if MPs isolated from the sputum of patients with pulmonary diseas, in particular CF and dyskinesia primary ciliar (DPC), contribute to induction of lung inflammation. Swiss CD11 mice (6–8 weeks old male) were induced by intratracheal administration of MPs isolated from the sputum of CF and DPC, LPS (20μg) or saline. Histologycal analysis was performed on H and E stained lung sections. In Bronchoalveolar lavage fluid (BALF) total cells were counted by trypan blue stain, whereas differential cell counts were performed on cytospin preparations stained with May-Grunwald Giemsa. LPS induced a significant increase of total cellular count as compared with controls. MPs obtained from CF patients in acute state were inflammatory as well, with a peak of total cell counts obtained with 100 x 106 MPs injected. Interestingly, MPs obtained from CF intermittent state were less pro-inflammatory in comparition to acute CF patients MPs, while DPC patients MPs determined an intermediate inflammatory levels, between those elicited by acute and intermittent CF MPs. The analysis of differential cell counts revealed that endotoxin, 50 and 100 x 106 MPs from Cfpatients, determined an increase in neutrophil numbers; no differences were found between acute and intemittent CF patients, whereas controls had only macrophages in their BALF. MPs from DPC patients have a different behaviour, since the most of inflammatory cells were macrophages. Our data suggest that MPs isolated from CF sputum could contribute to lung injury resultant in increase of total inflammatory cells and neutrophil recruitment.

A new biological marker isolated and characterizated in sputum from lung pathology patients: microparticles

Sputum is recognized as a very useful sampling method in Cystic Fibrosis (CF) for both research and clinical use aiding both the diagnosis and monitoring of lung disease inflammatory status. A great advantage of the technique is that it enables sampling of the airways in a non-invasive manner. We investigated on the presence and phenotype of microparticles (MPs) in the sputum, of acute and intermittent CF and Primary Ciliary Dyskinesia (PCD) adult patients. MPs are membrane vesicles that are released during cell activation and apoptosis. MPs have different biological effects depending on the cell from they originate. CF lung disease is characterized by massive neutrophil influx in the airways, their activation and eventually apoptosis. Spontaneous sputum, obtained from 21 CF patients (10 acute and 11 intermittent) and 7 patients with PCD, was liquefied with Sputasol. MPs were counted, visualized by electron microscopy, identified in the supernatants of treated sputum by cytofluorimetry and immunodecoration for leukocyte (CD11a), granulocyte (CD66b), and monocyte-macrophage (CD11b) antigens. Electron microscopy revealed that sputum MPs were in the 100-500 nm range and did not contain bacteria. CF sputa contained higher number of MPs in comparison with PCD sputa. CD66b and CD11a, but not CD11b, levels in MPs were significantly higher in CF than in PCD. Acute and intermittent patients presented significantly higher levels of CD11aand CD66b-expressing MPs respect to PCD patients. MPs are detectable in sputa obtained from CF patients and are predominantly of granulocyte origin. These findings are consistent with massive influx of neutrophils into CF airways and their accumulation on the surface of the airway epithelium. In this environment, neutrophils are activated by bacterial products, pro-inflammatory cytokines, and chemokines. Neutrophils undergo apoptosis, as normally happens in acute inflammation, but also postapoptotic necrosis, releasing toxic enzymes and oxygen radicals. MPs in CF sputa likely reflect both activation and apoptosis of neutrophils. The significantly less presence of granulocyte-derived MPs in PCD respect to CF patients, may be due to the fact that lung disease in PCD, although similar, is delayed in time as compared with CF. In CF airways, neutrophil-derived MPs could contribute to self-perpetuating inflammatory cycle, and may account for the exaggerated pro-inflammatory response of cells in CF patients. The MPs isolated from sputum could be a new non invasive methods and opens a new opportunity for the study of lung disease.