Browsing by Author "Oliveira, Ana"
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- Accuracy of faecal calprotectin and neutrophil Gelatinase B-associated Lipocalin in evaluating subclinical inflammation in UlceRaTIVE colitis-the ACERTIVE studyPublication . Magro, Fernando; Lopes, Susana; Coelho, Rosa; Cotter, Jose; Castro, Francisca Dias de; Sousa, Helena Tavares; Salgado, Marta; Andrade, Patrícia; Vieira, Ana Isabel; Figueiredo, Pedro; Caldeira, Paulo; Sousa, A.; Duarte, Maria A.; Avila, Filipa; Silva, João; Moleiro, Joana; Mendes, Sofia; Giestas, Silvia; Ministro, Paula; Sousa, Paula; Gonçalves, Raquel; Gonçalves, Bruno; Oliveira, Ana; Chagas, Cristina; Torres, Joana; Dias, Claudia Camila; Lopes, Joanne; Borralho, Paula; Afonso, Joana; Geboes, Karel; Carneiro, FátimaBackground and Aims: Mucosal healing and histological remission are different targets for patients with ulcerative colitis, but both rely on an invasive endoscopic procedure. This study aimed to assess faecal calprotectin and neutrophil gelatinase B-associated lipocalin as biomarkers for disease activity in asymptomatic ulcerative colitis patients. Methods: This was a multicentric cross-sectional study including 371 patients, who were classified according to their endoscopic and histological scores. These results were evaluated alongside the faecal levels of both biomarkers. Results: Macroscopic lesions [i.e. endoscopic Mayo score >= 1] were present in 28% of the patients, and 9% had active disease according to fht Ulcerative Colitis Endoscopic Index of Severity. Moreover, 21% presented with histological inflammation according to the Geboes index, whereas 15% and 5% presented with focal and diffuse basal plasmacytosis, respectively. The faecal levels of calprotectin and neutrophil gelatinase B-associated lipocalin were statistically higher for patients with endoscopic lesions and histological activity. A receiver operating characteristic-based analysis revealed that both biomarkers were able to indicate mucosal healing and histological remission with an acceptable probability, and cut-off levels of 150-250 mu g/g for faecal calprotectin and 12 mu g/g for neutrophil gelatinase B-associated lipocalin were proposed. Conclusions: Faecal calprotectin and neutrophil gelatinase B-associated lipocalin levels are a valuable addition for assessment of disease activity in asymptomatic ulcerative colitis patients. Biological levels of the analysed biomarkers below the proposed thresholds can rule out the presence of macroscopic and microscopic lesions with a probability of 75-93%. However, caution should be applied whenever interpreting positive results, as these biomarkers present consistently low positive predictive values.
- Antibacterial activity of propolis extracts from the south of PortugalPublication . Oliveira, Ana; Ferreira, Ana Luisa; Nunes, Susana; Dandlen, Susana; Miguel, Maria; Faleiro, Maria LeonorTo examine the antibacterial activity of diverse extracts of propolis harvested at winter and spring from several locations of Algarve, Portugal, against Gram-negative and Gram-positive bacteria was the main goal of the present work. For such, the antibacterial activity was determined by agar diffusion. The results showed that all tested bacterial strains showed susceptibility to diluted propolis extracts and in a dose-dependent manner. Two propolis samples collected at springtime showed higher antibacterial activity, in comparison with samples harvested at wintertime. Ethanolic and methanolic extracts have a very similar activity (P<0.05). Helicobacter pylori strains J99 and 26695 were the most susceptible strains to the tested extracts (33.67 +/- 2.52 mm and 35.67 +/- 0.58mm, respectively). This study constitutes the first approach of the biological activities of Portuguese propolis from the Algarve region and evidences its potential use to combat bacterial infections, in particular against the gastric pathogen H. pylori.
- Chitosan-Hyaluronic acid hybrid vectors for retinal gene therapyPublication . Oliveira, Ana; Bitoque, Diogo; Silva, Gabriela A.
- Combining hyaluronic acid with chitosan enhances gene deliveryPublication . Oliveira, Ana; Bitoque, Diogo; Silva, GabrielaThe low gene transfer efficiency of chitosan-DNA polyplexes is a consequence of their high stability and consequent slow DNA release. The incorporation of an anionic polymer is believed to loosen chitosan interactions with DNA and thus promote higher transfection efficiencies. In this work, several formulations of chitosan-DNA polyplexes incorporating hyaluronic acid were prepared and characterized for their gene transfection efficiency on both HEK293 and retinal pigment epithelial cells. The different polyplex formulations showed morphology, size, and charge compatible with a role in gene delivery. The incorporation of hyaluronic acid rendered the formulations less stable, as was the goal, but it did not affect the loading and protection of the DNA. Compared with chitosan alone, the transfection efficiency had a 4-fold improvement, which was attributed to the presence of hyaluronic acid. Overall, our hybrid chitosan-hyaluronic acid polyplexes showed a significant improvement of the efficiency of chitosan-based nonviral vectors in vitro, suggesting that this strategy can further improve the transfection efficiency of nonviral vectors.
- Comparing the continuous geboes score with the Robarts Histopathology Index: definitions of histological remission and response and their relation to faecal calprotectin levelsPublication . Magro, Fernando; Lopes, Joanne; Borralho, Paula; Lopes, Susana; Coelho, Rosa; Cotter, Jose; de Castro, Francisca Dias; Sousa, Helena Tavares; Salgado, Marta; Andrade, Patricia; Vieira, Ana Isabel; Figueiredo, Pedro; Caldeira, Paulo; Sousa, A.; Duarte, Maria A.; Avila, Filipa; Silva, Joao; Moleiro, Joana; Mendes, Sofia; Giestas, Silvia; Ministro, Paula; Sousa, Paula; Goncalves, Raquel; Goncalves, Bruno; Oliveira, Ana; Chagas, Cristina; Cravo, Marilia; Dias, Claudia Camila; Afonso, Joana; Portela, Francisco; Santiago, Mafalda; Geboes, Karel; Carneiro, FatimaBackground and Aims: The histological status of ulcerative colitis [UC] patients in clinical and endoscopic remission has gained space as an important prognostic marker and a key component of disease monitoring. Our main aims were to compare two histological indexes-the continuous Geboes score [GS] and the Robarts Histopathology index [RHI]-regarding their definitions of histological remission and response, and the ability of faecal calprotectin [FC] levels to discriminate between these statuses. Methods: This was an analysis of three prospective cohorts including 422 patients previously enrolled in other studies. Results: The two continuous scores [GS and RHI] were shown to be significantly correlated [correlation coefficient of 0.806, p < 0.001] and particularly close regarding their definition of histological response: 95% and 88% of all patients classified as having/not having [respectively] histological response according to RHI also did so according to GS. Moreover, median FC levels in patients with histological response were lower than those in patients without histological response [GS: 73.00 vs 525.00, p < 0.001; RHI: 73.50 vs 510.00, p < 0.001]; a similar trend was observed when FC levels of patients in histological remission were compared to those of patients with histological activity [GS: 76.00 vs 228.00, p < 0.001; RHI: 73.50 vs 467.00, p < 0.001]. FC levels allowed us to exclude the absence of histological remission [according to RHI] and absence of histological response [according to RHI and GS], with negative predictive values varying from 82% to 96%. However, optimization of the FC cut-off to exclude the absence of histological remission, as for the continuous GS, falls within values that resemble those of the healthy population. Conclusion: The continuous GS and RHI histological scores are strongly correlated in their definitions of histological response. An absence of histological remission could only be excluded at physiological levels of FC.
- Comparison of different histological indexes in the assessment of UC activity and their accuracy regarding endoscopic outcomes and faecal calprotectin levelsPublication . Magro, Fernando; Lopes, Joanne; Borralho, Paula; Lopes, Susana; Coelho, Rosa; Cotter, Jose; de Castro, Francisca Dias; Sousa, Helena Tavares; Salgado, Marta; Andrade, Patricia; Vieira, Ana Isabel; Figueiredo, Pedro; Caldeira, Paulo; Sousa, A.; Duarte, Maria A.; Avila, Filipa; Silva, Joao; Moleiro, Joana; Mendes, Sofia; Giestas, Silvia; Ministro, Paula; Sousa, Paula; Goncalves, Raquel; Goncalves, Bruno; Oliveira, Ana; Rosa, Isadora; Rodrigues, Marta; Chagas, Cristina; Dias, Claudia Camila; Afonso, Joana; Geboes, Karel; Carneiro, FatimaObjective Histological remission is being increasingly acknowledged as a therapeutic endpoint in patients with UC. The work hereafter described aimed to evaluate the concordance between three histological classification systems-Geboes Score (GS), Nancy Index (NI) and RobartsHistopathologyIndex (RHI), as well as to evaluate their association with the endoscopic outcomes and the faecal calprotectin (FC) levels. Design Biopsy samples from 377 patients with UC were blindly evaluated using GS, NI and RHI. The results were compared with the patients' Mayo Endoscopic Score and FC levels. Result GS, NI and RHI have a good concordance concerning the distinction between patients in histological remission or activity. RHI was particularly close to NI, with 100% of all patients classified as being in remission with NI being identified as such with RHI and 100% of all patients classified as having activity with RHI being identified as such with NI. These scores could also predict the Mayo Endoscopic Score and the FC levels, with their sensitivity and specificity levels depending on the chosen cut-offs. Moreover, higher FC levels were statistically associated with the presence of neutrophils in the epithelium, as well as with ulceration or erosion of the intestinal mucosa. Conclusions GS, NI and RHI histopathological scoring systems are comparable in what concerns patients' stratification into histological remission/activity. Additionally, FC levels are increased when neutrophils are present in the epithelium and the intestinal mucosa has erosions or ulcers. The presence of neutrophils in the epithelium is, indeed, the main marker of histological activity.
- Development and validation of risk matrices for Crohn's Disease outcomes in patients who underwent early therapeutic interventionsPublication . Dias, Cláudia Camila; Rodrigues, Pedro Pereira; Coelho, Rosa; Santos, Paula Moura; Fernandes, Samuel; Lago, Paula; Caetano, Cidalina; Rodrigues, Angela; Portela, Francisco; Oliveira, Ana; Ministro, Paula; Cancela, Eugenia; Vieira, Ana Isabel; Barosa, Rita; Cotter, Jose; Carvalho, Pedro; Cremers, Isabelle; Trabulo, Daniel; Caldeira, Paulo; Antunes, Artur; Rosa, Isadora; Moleiro, Joana; Peixe, Paula; Herculano, Rita; Gonçalves, Raquel; Gonçalves, Bruno; Sousa, Helena Tavares; Contente, Luis; Morna, Henrique; Lopes, Susana; Magro, FernandoIntroduction: The establishment of prognostic models for Crohn's disease [CD] is highly desirable, as they have the potential to guide physicians in the decision-making process concerning therapeutic choices, thus improving patients' health and quality of life. Our aim was to derive models for disabling CD and reoperation based solely on clinical/demographic data. Methods: A multicentric and retrospectively enrolled cohort of CD patients, subject to early surgery or immunosuppression, was analysed in order to build Bayesian network models and risk matrices. The final results were validated internally and with a multicentric and prospectively enrolled cohort. Results: The derivation cohort included a total of 489 CD patients [64% with disabling disease and 18% who needed reoperation], while the validation cohort included 129 CD patients with similar outcome proportions. The Bayesian models achieved an area under the curve of 78% for disabling disease and 86% for reoperation. Age at diagnosis, perianal disease, disease aggressiveness and early therapeutic decisions were found to be significant factors, and were used to construct user-friendly matrices depicting the probability of each outcome in patients with various combinations of these factors. The matrices exhibit good performance for the most important criteria: disabling disease positive post-test odds = 8.00 [2.72-23.44] and reoperation negative post-test odds = 0.02 [0.00-0.11]. Conclusions: Clinical and demographical risk factors for disabling CD and reoperation were determined and their impact was quantified by means of risk matrices, which are applicable as bedside clinical tools that can help physicians during therapeutic decisions in early disease management.
- Human-derived NLS enhance the gene transfer efficiency of chitosanPublication . Bitoque, Diogo; Morais, Joana; Oliveira, Ana; Sequeira, Raquel L.; Calado, Sofia; Fortunato, Tiago M.; Simão, Sónia; Rosa Da Costa, Ana; Silva, Gabriela A.Nuclear import is considered as one of the major limitations for non-viral gene delivery systems and the incorporation of nuclear localization signals (NLS) that mediate nuclear intake can be used as a strategy to enhance internalization of exogenous DNA. In this work, human-derived endogenous NLS peptides based on insulin growth factor binding proteins (IGFBP), namely IGFBP-3 and IGFBP-5, were tested for their ability to improve nuclear translocation of genetic material by non-viral vectors. Several strategies were tested to determine their effect on chitosan mediated transfection efficiency: co-administration with polyplexes, co-complexation at the time of polyplex formation, and covalent ligation to chitosan. Our results show that co-complexation and covalent ligation of the NLS peptide derived from IGFBP-3 to chitosan polyplexes yields a 2-fold increase in transfection efficiency, which was not observed for NLS peptide derived from IGFBP-5. These results indicate that the integration of IGFBP-NLS-3 peptides into polyplexes has potential as a strategy to enhance the efficiency of non-viral vectors.
- Impact of early surgery and immunosuppression on Crohn's disease disabling outcomesPublication . Magro, Fernando; Dias, Cláudia C.; Coelho, Rosa; Santos, Paula M.; Fernandes, Samuel; Caetano, Cidalina; Rodrigues, Angela; Portela, Francisco; Oliveira, Ana; Ministro, Paula; Cancela, Eugenia; Vieira, Ana I.; Barosa, Rita; Cotter, Jose; Carvalho, Pedro; Cremers, Isabelle; Trabulo, Daniel; Caldeira, Paulo; Antunes, Artur; Rosa, Isadora; Moleiro, Joana; Peixe, Paula; Herculano, Rita; Gonçalves, Raquel; Gonçalves, Bruno; Sousa, Helena Tavares; Contente, Luis; Morna, Henrique; Lopes, SusanaBackground and Aims: The definition of early therapeutic strategies to control Crohn's disease aggressiveness and prevent recurrence is key to improve clinical practice. This study explores the impact of early surgery and immunosuppression onset in the occurrence of disabling outcomes. Methods: This was a multicentric and retrospective study with 754 patients with Crohn's disease, who were stratified according to the need for an early surgery (group S) or not (group I) and further divided according to the time elapsed from the beginning of the follow-up to the start of immunosuppression therapy. Results: The rate of disabling events was similar in both groups (S: 77% versus I: 76%, P = 0.700). The percentage of patients who needed surgery after or during immunosuppression therapy was higher among group S, both for first surgeries after the index event (38% of groups S versus 21% of group I, P, 0.001) and for reoperations (38% of groups S versus 12% of group I, P < 0.001). The time elapsed to reoperation was shorter in group I (HR = 2.340 [1.367-4.005]), stratified for the onset of immunosuppression. Moreover, reoperation was far more common among patients who had a late start of immunosuppression (S-36: 50% versus S0-6: 27% and S6-36: 25%, P < 0.001) and (I-36: 16% versus I0-6: 5% and I6-36: 7%, P, 0.001). Conclusions: Although neither early surgery nor immunosuppression seem to be able to prevent global disabling disease, an early start of immunosuppression by itself is associated with fewer surgeries and should be considered in daily practice as a preventive strategy.
- Sustained gene expression in the retina by improved episomal vectorsPublication . Calado, Sofia; Oliveira, Ana; Machado, Susana; Haase, Rudolf; Silva, GabrielaGene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy-low levels of transgene expression, vector loss during mitosis, and gene silencing-need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.