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- Variants of the PKLR gene are associated with rates of hospitalization in Angolan children with sickle cell anemiaPublication . Manco, Licínio; Santos, Brígida; Faustino, Paula; Ginete, Catarina; Brito, Miguel; Arez, Ana PaulaBackground: Sickle cell anemia (SCA), caused by the c.20 A > T (p.Glu6Val) mutation in the HBB gene, is one of the most prevalent hereditary diseases in Sub-Saharan Africa. Patients with the HbSS genotype exhibit variable phenotypic expressions and disease severity, often reflected in hospitalization rates. Reduced activity of pyruvate kinase (PKR, gene: PKLR), a key enzyme in glycolysis, impacts SCA pathophysiology through 2,3-DPG accumulation and ATP deficit. This study analyzed the association of three PKLR variants with hospitalization and clinical parameters in Angolan children with SCA. Material and methods: Sixty-three SCA children (3–12 years) were monitored in a prospective study (2019–2022). Polymorphisms rs8177970 and rs3020781 (intron 3) and rs1052177 (3' UTR) in the PKLR gene were analyzed via PCR-RFLP. Allele frequencies and Hardy-Weinberg equilibrium (HWE) were assessed using Arlequin 3.5, and SPSS 27 was used for statistical analyses. Association studies were performed using the Mann-Whitney U test, combining heterozygous and homozygous individuals for the minor allele into a single group. Results: Minor allele frequencies for rs8177970, rs3020781, and rs1052177 were 0.214, 0.221, and 0.377, respectively, with all genotype distributions consistent with the HWE. Polymorphism rs1052177 was significantly associated with hospitalization rates (p = 0.025), while rs8177970 showed a near-significant association (p = 0.068). No associations were found with hematological parameters. Conclusion: This study, conducted in SCA children from Angola, found potential links between two PKLR SNPs and hospitalization rates. The PKLR gene may act as a genetic modifier of the clinical progression of SCA, as these SNPs can affect gene expression levels.
- Refractory status epilepticus outcome: a retrospective study from a Portuguese hospitalPublication . Palhete-Ferreira, Lígia; Dias, J.; Peralta, A. R.; Bentes, C.Purpose: Refractory Status Epilepticus (RSE) is a severe neurological emergency characterized by refractoriness to initial anti-seizure medications (ASM) and often requiring intravenous anesthetics therapy (IVAT) in an intensive care unit (ICU) setting. Despite previous efforts, biomarkers of successful RSE reversal are still lacking. Aim: study clinical predictors of successful IVAT weaning off and outcome in patients with RSE under IVAT. Method: Retrospective study including RSE adult patients, under optimized IVAT for at least 72h, between November 2017 and December 2018. Clinical and functional outcome (mRS) data were collected at ICU discharge and at the last follow-up time. The association between clinical variables, successful IVAT weaning-off, and functional outcome was analyzed using statistical methods. Results: 32 patients (mean age 67.6 years; mean time follow-up 17months) were included, and 58 IVAT weaning-off attempts were analyzed, of which 41,3% were successful. All patients with a previous epilepsy diagnosis had a successful IVAT weaning-off attempt (p=0,022). However, no clinical factor was found to be an independent predictor of successful IVAT weaning-off in our binary-logistic regression model. At ICU discharge, 96,6% patients had mRS >2, 36,2% had died, and there was a positive association between mRS score and age (p=0,000), RSE type (p=0,004; mostly focal-motor RSE) and etiology (p=0,006), number of ASMs (p=0,004), treatment with LCS (p=0,000) and VPA (p=0,004) and STESS score (p=0,000). At last follow-up, 90,9% patients had mRS >2, 86,4% had died, and there was an association between mRS score and RSE etiology (p=0,049; mostly acute-symptomatic) and CNS infection (p=0,022). Conclusion: This study shows a negative outcome associated with RSE. Successful IVAT weaning-off was more frequent in epilepsy patients. RSE patients’ functional outcome was associated with multiple clinical factors, although identification of independent predictors was not possible. Larger, prospective, and multimodal studies are needed to better understand refractory forms of RSE and outcome predictors.
- Circulating miR-146a and mir-134 as potential therapeutic biomarkers in refractory status epilepticus: an exploratory studyPublication . Palhete-Ferreira, Lígia; Leal, B.; Santos, C.; Franco, S.; Parreira, S.; Peralta, A. R.; Moniz, I.; Gaibino, N.; Teixeira, A.; Mateus, C.; Santos, R.; Bentes, C.Purpose: Refractory Status Epilepticus (RSE) is defined by persisting electroclinical/electrographic seizures despite intravenous therapy with two anti-seizure medication (ASM), including a benzodiazepine. The therapeutic approach includes underlying cause treatment, other ASMs and intravenous anesthetic therapy (IVAT). In clinical practice, biomarkers for RSE treatment are needed, aiming for its effective reversal and diligent IVAT reduction. MicroRNAs (miR) have been identified in epileptogenesis, including refractory epilepsies, with promising roles as diagnostic, prognostic and therapeutic response biomarkers. Previous studies, animal model of RSE and SE patients, reported miR upregulation. However, serum miR in RSE clinical model have not been studied. We aim to evaluate circulating levels of miR-146a and mir-134 in patients under IVAT for RSE treatment. Method: MiR-146a and miR-134 were quantified by real-time PCR in serum of adult patients with optimized IVAT for RSE treatment (EEG validated) and healthy individuals. Anoxic etiology was excluded. Relative expression values were calculated using 2-ΔΔCT method and compared with controls (Mann-Whitney and T-Student, p<0.05). Results: MiR circulating levels were quantified in 19 patients (8.53±15.70 years), 84,21% with electroclinical status epilepticus and 15,79% with electrographic status epilepticus, and 10 healthy individuals (27.60±12.00 years). RSE patients with optimized IVAT dose presented downregulated serum levels of miR-146a (20-fold lower (p<0,001) and miR-134 (3-fold lower,p<0,001). Conclusion: In this work, a significant downregulation in serum levels of miR-146a and miR-134 was demonstrated in optimized IVAT for RSE treatment, contradicting miR upregulation stated in SE, both in animal and human models. We postulate that our results are due to the therapy established for RSE, reflecting the importance of intervention models in mechanisms of neuroinflammation, neuronal excitability and dendritic spine morphology. Future work should analyze the role of miRs as RSE therapeutical response biomarkers.
- Analysis of the pilot study of the International Hemoglobinopathy Research Network (INHERENT)Publication . Kountouris, Petros; Stephanou, Coralea; Xenophontos, Maria; Minaidou, Anna; Christou, Sotiroula; Savvidou, Iren; Rekleiti, Anna; Pyrovolaki, Katerina; Constantinou, Natasa; Tshilolo, Leon; Nzengu, F.; Fazili, S.; Brito, MiguelIntroduction: Hemoglobinopathies, including sickle cell disease (SCD) and thalassemia syndromes, represent the commonest monogenic diseases. Although their pathogenesis is well established, the diverse clinical manifestations and their varying degree of severity are less understood and are partly influenced by genetic modifiers. Despite the identification and characterization of genetic modifiers by previous studies, these are, as yet, insufficient to guide treatment recommendations or stratify patients reliably. The International Hemoglobinopathy Research Network (INHERENT) investigates the role of genetic modifiers in hemoglobinopathies, intending to identify and validate further disease modifiers. This pilot study tested the operational feasibility of the INHERENT study across different geographic and healthcare settings and identified and addressed challenges in performing a large, multi-ethnic genome-wide association study (GWAS). Methods: The following steps of the study implementation have been tested: (a) obtaining local bioethics approval based on the applicable local legal framework, (b) patient enrolment, written informed consent, and data collection using a common case report form (CRF), (c) sample collection and shipment, (d) genotyping of globin genes, (e) centralized GWAS experiments, and (f) statistical analysis. The completeness of the collected dataset was also assessed. Results: The pilot study enrolled 1044 patients from 15 centres spanning 8 countries, namely Angola, Cyprus (2), Denmark, DR Congo, Greece (3), Malaysia (3), Nigeria (3), and the USA. An additional 13 centres have obtained bioethics approval, but have not initiated patient enrolment yet. The distribution by disease group is 42.2% SCD and 57.8% thalassemia patients, while the median age is 28 years (mean: 31.1), with 69.6% adult and 30.4% pediatric patients. Data completeness (affirming presence, absence, or not enough data) of key parameters related to medical complications is approximately 85%, while the range of completeness for laboratory parameters was wide, with a maximum at 80%. Notably, a higher rate of cardiac/pulmonary, kidney/liver, endocrinological, and bone complications is observed in adult thalassemia patients, while a higher rate of pain-related and acute anemia complications is observed in pediatric SCD patients. Biological material for 768 of the patients was shared centrally, and GWAS experiments have been performed using the Illumina GSA SNP array. Key challenges identified in the pilot study include: a) the unavailability of key phenotypic data in routine clinical practice in several countries, particularly when the tests are not covered by insurance; b) the need for a more detailed standardization and simplification of the INHERENT CRF to ensure uniform and consistent collection of data across participating centers; c) the unavailability, limited access or high costs of molecular diagnosis services in some countries; d) varying levels of knowledge and technical skills in laboratory work across centers; e) challenges related to the storage, quality and shipping of biological material in some countries. Conclusion: The pilot study tested common standards developed within INHERENT and enabled early identification of key challenges associated with the execution of a large, multi-ethnic study for hemoglobinopathies. The pilot is pivotal for scaling up the INHERENT GWAS across the entire network, enabling the study of a hemoglobinopathy population of unprecedented size and diversity.
- Translating research into practice: implementing the LEARNER SCD pregnancy study in AngolaPublication . Ginete, Catarina; Brito, Miguel; Gomes, Tatiana; Pitangueira, Helena; Mendes, Manuela; Furtado, Ana; Alves, Lígia; Simão, Fernanda; Gonçalves, Mauer; Morais, JoanaIntroduction: Pregnancy in Sickle Cell Disease (SCD), a severe genetic condition highly prevalent in Sub-Saharan Africa, is usually associated with an increase in severe outcomes. Not only do the common symptoms of the disease, such as severe anemia and vaso-occclusive crises, tend to exacerbate, but also the risk of eclampsia, pre-eclampsia, maternal and fetal death, intrauterine growth restrictions, and low birth weight is higher. Medical surveillance during pregnancy is essential, and also, the search for prophylactic and affordable measures is an urgent need, especially in Low- and Middle-income countries with high prevalence of this disease. The use of daily low-dose aspirin is considered safe in pregnant women with SCD and is recommended after 12 weeks of gestational age by the British Society of Haematology for those at severe risk of pre-eclampsia. The LEARNER clinical study (NCT06417411) aims to evaluate the effects of daily low-dose aspirin in SCD pregnancy, comparing its impact on severe outcomes if this prophylactic and affordable medication is started in the first or the second trimester. Methods: This study aims to recruit 450 pregnant women with a confirmed SCD diagnosis in multiple maternity and infant hospitals in Luanda, Angola. Consenting patients will be assigned to the first (weeks 6–13) or second (weeks 14–27) trimester groups according to their gestational age, as confirmed by ultrasound. Participants will start daily low-dose aspirin and will do regular follow-up appointments till 6 weeks postpartum. Aspirin will be interrupted at week 36, delivery time, or earlier if decided by the clinical team. Results: Recruitment started in March 2024, after ethical and regulatory approvals (Parecer no. 52/CEMS/2023, and 99/ARMED/MINSA/2024). In 15 months, 113 women were enrolled in the study, 57 in the first trimester and 56 in the second trimester. To date, 271 severe events, 3 associated with the medication, 9 cases of pre-eclampsia/eclampsia, 36 preterm deliveries, 5 miscarriages, 1 perinatal death, and 4 maternal deaths were registered. Conclusion: The current sample size is too small to draw statistically significant conclusions about the efficacy of starting low-dose daily aspirin earlier in pregnancy. Although the project has been promoted in hospitals and health centers through the media, in newspapers, and on television, to increase the participation in the study, the number of enrolled patients is below the expected, as most pregnant women tend to seek hospital care only during the later stages of pregnancy. It is urgent to invest in health literacy and SCD education in Angola and increase patients' awareness of the need to do prenatal and follow-up consultations during pregnancy for the prevention of pregnancy-associated complications in women with SCD.
- Frequency, characteristics and predictors oc central and peripheral neurological involvement in Sjögren’s disease: data from PORTRESS, the Portuguese registry of Sjögren’s diseasePublication . Costa, R. Pereira da; Bandeira, Matilde; Silvério-António, Manuel; Lopes, Ana Rita; Santos, Filipe Cunha; Pereira, Paulo J.; Raimundo, Diana Belchior; Cunha, Anita; Oliveira, Cláudia Pinto; Duarte, Ana Catarina; Dias, J. Madruga; Santos, Mariana Emília; Gonçalves, Maria João; Moniz, Ana Catarina; Maduro, Ana Isabel; Luis, Mariana; Valido, Ana; Oliveira, Margarida; Brites, Luísa; Tenazinha, Catarina; Khmelinskii, Nikita; Barcelos, Filipe; Fonseca, João Eurico; Romão, Vasco C.Background: Sjogren's disease (SjD) systemic affection is being increasingly recognized. The prevalence of peripheral (PNS) and central (CNS) nervous system involvement in SjD and risk factors for these manifestations have not been clearly established. Objectives: To define the clinical characteristics of SjD patients with CNS and/or PNS involvement and to find predictors of this involvement.
- Cardiotoxicidade: avaliação do strain da aurícula esquerda por ETTPublication . Sousa, Rui; Silva, Joana; Henriques, Madalena; Cavaco, Sofia; Clérigo, Anália Matos; Fonseca, VirgíniaIntrodução: O cancro da mama é uma das neoplasias malignas mais prevalentes, sendo a cardiotoxicidade induzida pelo tratamento com antraciclinas e trastuzumab uma complicação importante, associada à disfunção miocárdica e à insuficiência cardíaca. O ecocardiograma transtorácico (ETT) é considerado o método gold standard na avaliação da função cardíaca, sendo a fração de ejeção do ventrículo esquerdo (FEVE) e o strain longitudinal global (GLS) os principais parâmetros utilizados para a deteção precoce de cardiotoxicidade. O strain da aurícula esquerda (AE) representa a deformação miocárdica desta estrutura e a sua avaliação é realizada com base em três parâmetros principais, entre os quais se destaca o peak atrial longitudinal strain (PALS) por melhor refletir a função global da AE. O strain da AE surge, deste modo, como um potencial marcador precoce de cardiotoxicidade induzida pela quimioterapia. Objetivo: Avaliar o strain da aurícula esquerda, por ETT, como preditor de cardiotoxicidade, em diferentes fases do tratamento com antraciclinas e trastuzumab, em mulheres com neoplasia da mama.
- O impacto da hipertensão arterial no desempenho cognitivoPublication . Fonseca, Virgínia; Camacho, Gabriela; Rocha, Mariana; Clérigo, Anália Matos; Guimarães, TeresaIntrodução: A hipertensão arterial é um fator de risco cardiovascular com elevada prevalência na população adulta. Para além dos seus efeitos sobre o sistema cardiovascular, pode interferir com os mecanismos homeostáticos cerebrovasculares, comprometendo a perfusão e o metabolismo cerebral, com repercussões negativas no desempenho cognitivo. Está particularmente associada a alterações nas funções executivas, frequentemente um dos primeiros domínios a serem afetados em contextos patológicos. Objetivo: Avaliar o desempenho cognitivo em indivíduos normotensos e hipertensos.
- Evaluating the impact of using natural fixative agents as alternatives to formaldehyde in histopathology: a systematic reviewPublication . Horta, Andreia; Condesso, Nádia; Maia-Matos, Mário; Borges-Ferro, AThe histological technique involves several steps to prepare tissues for microscopic analysis, with fixation being one of the key steps. Formaldehyde is the most used fixing agent; however, it poses significant health risks and is classified as a Group 1 human carcinogen by the IARC and the NTP. This concern has prompted the search for safer alternatives. This study aims to identify a less harmful natural substitute for formaldehyde that can reduce or eliminate exposure to this hazardous reagent. To achieve this objective, a systematic review explored the potential of natural fixatives that can perform similar functions with lower toxicity.
- Antibody production for diagnostic applications and therapeutic development: is it possible to reduce the use of animals?Publication . Sushchenko, Yelyzaveta; Turiel, Ema; Aguiar, Joana; Janeiro, Mariana; Borges-Ferro, ATraditionally, antibodies have been produced using the hybridoma technique, which involves immunization and the sacrifice of animals. Although this has revolutionized the practice of “traditional” immunohistochemistry, it currently raises some ethical questions regarding animal suffering. Therefore, it is important to develop methods that are viable to obtain quality antibodies for human diagnosis and therapy, while also having a less significant impact on animal welfare. There are several promising methods, such as the use of mammalian cells, the expression of antibodies in phage display, and even in yeasts. This review aims to explore alternative methods for antibody production, highlighting their advantages and limitations.