Olga Lourenço

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

As common microbiological methods for the assessment of bacteriostatic or bactericidal activities are very time-consuming, in this work we describe that the use of a novel far-red fluorescent stain, Vybrant DyeCycle Ruby (DCR) for the flow cytometric analysis of fluoroquinolone (ciprofloxacin) bacteriostatic and bactericidal activities in Escherichia coli proved to be specific for bacterial DNA and, after ciprofloxacin exposure, DNA distribution analysis was achieved using a 7.5 μM DCR concentration to stain 5 × 105 ethanol-fixed bacterial cells. The analysis of the bacterial DNA histograms obtained from the ciprofloxacin concentrations tested, enabled the distinction between ciprofloxacin bacteriostatic and bactericidal activities.

Serum biomarkers in elderly asthma

Abstract Objective: Asthma is usually misdiagnosed and under-treated in the elderly population, resulting in complications and increased severity to the patient. In this review, we describe some of the most important serum markers of asthma studied so far, reporting their outcomes and possible prediction of asthma in the elderly population. Methods: The PubMed electronic database was used to search for promising serum biomarkers of asthma studied in original articles published in peer-reviewed journals from 2000 to January 2013. Results: A total of 13 relevant serum biomarkers were selected, including IgE, CRP, high sensitive CRP, IL-6, IL-8, IL-17, TNF-α, neopterin, serum amyloid A, eosinophil cationic protein, leukolysin, YKL-40 and soluble CD86. Conclusions: Although the major focus of treatment and research has been on allergic asthma, several forms of the disease are recognized, such as neutrophilic asthma, which is characteristic of older patients. Different phenotypes imply different treatments and so it becomes important to correctly determine which type of asthma the patient is suffering from. Serum markers capable of supporting a diagnosis of asthma are needed in order to counter mistreatment and misdiagnosis with other obstructive airways disease (OAD) in elderly patients. As convenient as serum markers may seem to be, a marker capable of accurately identifying asthma with sufficient specificity is yet to be found.

T cells in sputum of asthmatic patients are activated independently of disease severity or control

BACKGROUND T cells play an important role in bronchial asthma. Although airway CD4+ T cells have been extensively studied previously, there are hardly any studies relating CD8+ T cell activation and disease symptoms. OBJECTIVES The aim of this study was to analyse the association between T cell activation in induced sputum T cells and asthma severity and control; and to evaluate T cell subpopulations in the same subgroups. METHODS Fifty allergic asthmatic patients were recruited and lung function testing was performed. Airway cells were obtained by sputum induction via inhalation of hypertonic saline solution. CD3, CD4, CD8, CD28, CD25 and CD69 were studied by flow cytometry in whole induced sputum and peripheral blood cells. RESULTS Total induced sputum T cells and CD8+ T cells had a higher relative percentage of the activation markers CD25 and CD69 in comparison with peripheral blood. In sputum, the relative percentage of CD25 was higher in CD4+ T cells when compared to CD8+ T cells and the reverse was true regarding CD69. However, neither disease severity nor control were associated with the relative percentage of CD25 or CD69 expression on T cells in sputum. CONCLUSIONS Both CD4+ and CD8+ T cells are activated in the lungs and peripheral blood of asthmatic patients. However, with the possible exception of CD69+ CD8+ T lymphocytes in the sputum, there is no association between T cell activation phenotype in the target organ and disease severity or control.

Polyazamacrocycles as Potential Antitumor Agents for Human Prostate Cancer Cells

Polyazamacrocycles are currently being studied and used in a variety of applications beyond their traditional place in supramolecular and co‐ordination chemistry. This study suggests additional applications of these compounds with particular emphasis on their use as antiproliferative agents that could be potentially used to treat cancer. Four polyazamacrocycles were tested in human prostate cancer LNCaP and prostate epithelial PNTA1 cells to analyze changes in cell proliferation and cell death capabilities. Their intracellular localization was also evaluated by confocal microscopy. The results show a decrease in proliferation rate and cell viability of LNCaP and PNTA1, after treatment with these compounds. The decrease in the number of viable cells is similar for the majority of the compounds studied, and at higher concentration, the proliferation efficiency decreased significantly in the cell lines studied. Also, our results suggest that L and L2 induce early apoptosis in PNTA1 cells and late apoptosis/necrosis in LNCaP cells. The compounds did not induce a significant increase in necrosis of both cell types. Although the compounds did not localize in a unique organelle, all of them have as main target the Golgi apparatus and other localization profiles differed depending on the cell line.

Demographic, laboratory and clinical characterisation of adult portuguese asthmatic patients

BACKGROUND Asthma is a heterogeneous chronic inflammatory condition characterised by reversible airway obstruction and hyperresponsiveness associated with underlying bronchial inflammation and structural changes. It represents an increasing health problem and is a huge burden on the patients, their families and society. The aim of the study was to characterise the adult asthmatic population attending a Hospital Allergy Clinic between the years of 2003 and 2006. METHODS Clinical files from the Allergy Outpatient Clinic of Cova da Beira Hospital were sequentially studied. The total population analysed included 335 female and 130 male asthmatic patients. Bronchial asthma was characterised by clinical history, skin prick testing to aeroallergens, determination of total and specific IgE and lung function testing, and classified according to international guidelines. RESULTS Of the patients studied, 70 % had allergic asthma, and 30 % had non-allergic asthma. When compared to allergic asthma, non-allergic asthma was more frequently associated with older age, perennial symptoms and female gender. More allergic than non-allergic asthma patients also had rhinitis and the reverse was true regarding drug allergy and oesophageal reflux. Grass pollen and mites were the major sensitisers for allergic asthmatics. The sensitisation profile was significantly different between urban- and rural-based asthmatic patients regarding tree pollen, fungi and moulds. CONCLUSIONS In this population, rhinitis was more frequently associated with allergic than with non-allergic asthma. The two types of asthma did not differ in clinical severity or changes in lung function. Sensitisation profiles were different between the urban and rural patients.

Functional and phenotypic characterization of CD8+CD28+ and CD28­ T cells in atopic individuals sensitized to Dermatophagoides pteronyssinus

BACKGROUND CD8+ T suppressor cells may play a role in immunoregulation. Recent studies have characterized this population by the lack of the CD28 molecule. These CD8+CD28 T cells differ phenotypically and functionally from CD8 + CD28 + T cells. Little is known about CD8 + CD28 cells in atopy. Our aim was to analyze the phenotype and functional properties of CD8 + CD28T cells in atopic and non-atopic individuals. METHODS Peripheral blood mononuclear cells (PBMC) were obtained after density gradient centrifugation. CD8 + CD28 and CD8 + CD28 + T cells were isolated using immunomagnetic beads. Relative percentages of these cells and expression of several phenotypic markers were analyzed by flow cytometry. Proliferation was assessed by thymidine incorporation in isolated populations and in co-cultures with PBMC using Dermatophagoides pteronyssinus as stimulus. Cytokine synthesis was evaluated in culture supernatants by cytometric bead array. RESULTS The relative percentages of CD8+CD28 T cells and their phenotypic expression in atopic and non-atopic volunteers were not significantly different. However, CD8 + CD28 T cells showed greater proliferation than did CD8+CD28+ T cells when stimulated with D. pteronyssinus, although cytokine synthesis patterns were similar. CD8+CD28 co-cultures with PBMC showed greater proliferation than CD8+CD28+ T cell co-cultures, but cytokine synthesis patterns were not different. CONCLUSIONS Our data confirm phenotypic and functional differences between CD28+ and CD28 T cells, irrespective of atopic status. Purified human CD8+CD28 T cells, freshly isolated from peripheral blood, do not have suppressor properties on allergen-specific proliferation or on cytokine synthesis in PBMC.

MASK 2017: ARIA digitally-enabled, integrated, person-centred care for rhinitis and asthma multimorbidity using real-world-evidence

AbstractmHealth, such as apps running on consumer smart devices is becoming increasingly popular and has the potential to profoundly affect healthcare and health outcomes. However, it may be disruptive and results achieved are not always reaching the goals. Allergic Rhinitis and its Impact on Asthma (ARIA) has evolved from a guideline using the best evidence-based approach to care pathways suited to real-life using mobile technology in allergic rhinitis (AR) and asthma multimorbidity. Patients largely use over-the-counter medications dispensed in pharmacies. Shared decision making centered around the patient and based on self-management should be the norm. Mobile Airways Sentinel networK (MASK), the Phase 3 ARIA initiative, is based on the freely available MASK app (the Allergy Diary, Android and iOS platforms). MASK is available in 16 languages and deployed in 23 countries. The present paper provides an overview of the methods used in MASK and the key results obtained to date. These include a novel phenotypic characterization of the patients, confirmation of the impact of allergic rhinitis on work productivity and treatment patterns in real life. Most patients appear to self-medicate, are often non-adherent and do not follow guidelines. Moreover, the Allergy Diary is able to distinguish between AR medications. The potential usefulness of MASK will be further explored by POLLAR (Impact of Air Pollution on Asthma and Rhinitis), a new Horizon 2020 project using the Allergy Diary.

Human CD8+ T Cells in Asthma: Possible Pathways and Roles for NK-Like Subtypes

Asthma affects approximately 300 million people worldwide and is the most common chronic lung disease, which usually is associated with bronchial inflammation. Most research has focused upon the role of CD4+ T cells, and relatively few studies have addressed the phenotypic and functional roles of CD8+ T cell types and subtypes. Human NK-like CD8+ T cells may involve cells that have been described as CD8+CD28−, CD8+CD28−CD57+, CD8+CD27−, or CD8+ effector memory (TEM) cells, among other. However, most of the data that are available regarding these various cell types were obtained in murine models did not thoroughly characterize these cells with phenotypically or functionally or did not involve asthma-related settings. Nevertheless, one may conceptualize three principal roles for human NK-like CD8+ T cells in asthma: disease-promoting, regulatory, and/or tissue repair. Although evidence for some of these roles is scarce, it is possible to extrapolate some data from overlapping or related CD8+ T cell phenotypes, with caution. Clearly, further research is warranted, namely in terms of thorough functional and phenotypic characterization of human NK-like CD8+ T cells in human asthma of varying severity.