Browsing by Author "Azevedo, Pilar"
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- Isolation of Rasamsonia argillacea species complex in a Cystic Fibrosis Adult Patient - First Case in PortugalPublication . Carvalho, Dinah; Sabino, Raquel; Veríssimo, Cristina; Simões, Helena; Azevedo, Pilar; Marques Lito, Luis; Melo Cristino, JoséObjectives: Cystic fibrosis (CF) is the most common monogenetic autosomal reces sive disease in the human population. An important fungal biota has been described in respiratory secretions of patients suffering from CF being Aspergillus fumigatus and Candida albicans the most common fungi found. We report the isolation, for the first time in Portugal, of the emerging fungal pathogen Rasamsonia argillacea species complex, from a respiratory sample of an adult patient with CF. Material & Methods: A 51-year-old male patient with heterozygous CF due to muatations ΔF508/P205S, is being followed in Cystic Fibrosis Reference Center for about 10 years. In the last years, he has been consistently colonized with Methicilin-susceptible Staphylococcus aureus, Pseudomonas aeruginosa and Aspergillus section Fumigati whereby is under chronic suppression therapy with two inhaled antibiotics. Recently, there has been a progressive clinical respiratory functional deterioration. In a periodic evaluation, a miccrobiology control sputum was requested. Sample was cultured, in parallel, for bacteriol ogy and mycology evaluation. Results: Apart from detection of S. aureus and Raoultella ornithinolytica, after 3-5 days of incubation at 37ºC, the cultures showed several cream-coloured colonies, powdery to velvety. Microscopic examination showed hyaline septate hyphae, Penicillium-like conid iophores with rough wall, ovoid to cylindrical phyalides with a narrow neck and cylin drical unicellular smooth-walled microconidia, arranged in unbranched basipetal chains arising from phialides. The isolate was identified as Rasamsonia argillacea species complex based on its mor phology and confirmed by MALDI-TOF mass spectrometry. As no septate hyphae were seen on direct examination, a new sample was requested to exclude extrinsic contamina tion. The second sample was inoculated as previously, confirming the persistent presence of Rasamsonia argillacea species complex in the sputum of this patient. The identification of this isolate was further confirmed by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA, showing 100% homology with sequences deposited on data bases. Antifungal susceptibility testing showed high minimal inhibitory concentrations (MIC) to almost all tested antifungals (posaconazole, voriconazole, amphotericin B) and low MIC to anidulafungin. The patient had no great exacerbation of his respiratory prob lems and the isolated fungus was interpreted as colonization being the patient under more frequent surveillance. Conclusion: Although colonization of the upper respiratory tract in CF patients by R. argillacea species complex has been described as an emerging situation, the role of these fungi in clinical or functional deterioration of the disease remains controversial. Indeed, data about its real prevalence in the CF population are lacking. However, taking into ac count the ability of this species to predominantly affect the lungs, to induce pneumonia and to disseminate to adjacent organs or even to the central nervous system (CNS) in imunocompromised patients, it is essential to promote its accurate identification that is often misidentified as Penicillium spp. or Paecilomyces species. Antifungal susceptibility testing should be performed for epidemiological purposes and to guide therapy, as Rasamsonia spp. usually presents a marked antifungal resistance profile
- Profiling the erythrocyte membrane proteome isolated from patients diagnosed with chronic obstructive pulmonary diseasePublication . Alexandre, Bruno; Charro, Nuno; Blonder, Yosip; Lopes, Carlos; Almeida, Antonio Bugalho; Veenstra, Timothy; Penque, Deborah; Azevedo, Pilar; Chan, K.C.; Prieto, D.A.; Issaq, H.Structural and metabolic alterations in erythrocytes play an important role in the pathophysiology of Chronic Obstructive Pulmonary Disease (COPD). Whether these dysfunctions are related to the modulation of erythrocyte membrane proteins in patients diagnosed with COPD remains to be determined. Herein, a comparative proteomic profiling of the erythrocyte membrane fraction isolated from peripheral blood of smokers diagnosed with COPD and smokers with no COPD was performed using differential 16O/18O stable isotope labeling. A total of 219 proteins were quantified as being significantly differentially expressed within the erythrocyte membrane proteomes of smokers with COPD and healthy smokers. Functional pathway analysis showed that the most enriched biofunctions were related to cell-to-cell signaling and interaction, hematological system development, immune response, oxidative stress and cytoskeleton. Chorein (VPS13A), a cytoskeleton related protein whose defects had been associated with the presence of cell membrane deformation of circulating erythrocytes was found to be down-regulated in the membrane fraction of erythrocytes obtained from COPD patients. Methemoglobin reductase (CYB5R3) was also found to be underexpressed in these cells, suggesting that COPD patients may be at higher risk for developing methemoglobinemia. This article is part of a Special Issue entitled: “Integrated omics— Functional applications to blood and blood therapeutics”.
- SELDI-TOF biomarker signatures for cystic fibrosis, asthma and chronic obstructive pulmonary diseasePublication . Gomes-Alves, Patrícia; Imrie, Margaret; Gray, Robert D.; Nogueira, Paulo; Ciordia, Sergio; Pacheco, Paula; Azevedo, Pilar; Lopes, Carlos; De Almeida, António Bugalho; Guardiano, Micaela; Porteous, David J.; Albard, Juan P.; Boyd, A. Christopher; Penque, DeborahOBJECTIVES: The aim of this work was to establish protein profiles in serum and nasal epithelial cells of cystic fibrosis individuals in comparison with controls, asthma and chronic obstructive pulmonary disease patients for specific biomarker signatures identification. DESIGN AND METHODS: Protein extracts were analyzed by Surface Enhanced Laser Desorption/Ionization Time-Of-Flight Mass-Spectrometry (SELDI-TOF-MS). RESULTS: The mass spectra revealed a set of peaks with differential expression in serum and nasal cells among the different groups studied, resulting into peak signatures representative/specific of each pathology. Logistic regressions were applied to those peaks; sensitivity, specificity, Youden's indexes and area under the curve (AUC) of the respective receiver operating characteristic (ROC) curves were compared. DISCUSSION: Multivariate analysis demonstrated that combination of peaks has a better predictive value than the individual ones. These protein signatures may serve as diagnostic/prognostic markers for the studied diseases with common clinical features, or as follow-up assessment markers of therapeutic interventions.
- Serum proteomics signature of Cystic Fibrosis patients: A complementary 2-DE and LC–MS/MS approachPublication . Charro, Nuno; Hood, Brian L.; Faria, Daniel; Pacheco, Paula; Azevedo, Pilar; Lopes, Carlos; Bugalho de Almeida, António; Couto, Francisco M.; Conrads, Thomas P.; Penque, DeborahComplementary 2D-PAGE and ‘shotgun’ LC–MS/MS approaches were combined to identify medium and low-abundant proteins in sera of Cystic Fibrosis (CF) patients (mild or severe pulmonary disease) in comparison with healthy CF-carrier and non-CF carrier individuals aiming to gain deeper insights into the pathogenesis of this multifactorial genetic disease. 78 differentially expressed spots were identified from 2D-PAGE proteome profiling yielding 28 identifications and postulating the existence of post-translation modifications (PTM). The ‘shotgun’ approach highlighted altered levels of proteins actively involved in CF: abnormal tissue/airway remodeling, protease/antiprotease imbalance, innate immune dysfunction, chronic inflammation, nutritional imbalance and Pseudomonas aeruginosa colonization. Members of the apolipoproteins family (VDBP, ApoA-I, and ApoB) presented gradually lower expression from non-CF to CF-carrier individuals and from those to CF patients, results validated by an independent assay. The multifunctional enzyme NDKB was identified only in the CF group and independently validated by WB. Its functions account for ion sensor in epithelial cells, pancreatic secretion, neutrophil-mediated inflammation and energy production, highlighting its physiological significance in the context of CF. Complementary proteomics-based approaches are reliable tools to reveal pathways and circulating proteins actively involved in a heterogeneous disease such as CF.