National Institute of Health Dr. Ricardo Jorge
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Scientific Council of INSA: supporting scientific research and technological development [editorial]
No full textUma das principais atribuições do Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA) é promover e desenvolver
atividades de investigação científica e desenvolvimento tecnológico (IC&DT) contribuindo para ganhos em saúde
pública.
O Conselho Científico (CC) do INSA é o órgão responsável pela apreciação e acompanhamento das atividades de IC&DT,
órgão de reflexão e debate das atividades científicas do INSA, e órgão de ligação entre a estrutura de IC&DT e demais órgãos do INSA. Compete ao CC do INSA emitir parecer sobre o orçamento, o plano e o relatório anual de atividades, assim
como exercer, em relação à carreira de investigação científica (CIC), as competências que lhe estão cometidas. Este
órgão pronuncia-se ainda sobre todas as questões que lhe sejam submetidas pelo Conselho Diretivo (CD) da Instituição. [...
Cyanobacteria and cyanotoxins in treated water: to know in order to prevent
No full textA monitorização da qualidade da água para consumo humano relativamente à presença de cianobactérias é uma das atribuições do Departamento de Saúde Ambiental do Instituto Nacional de Saúde. A proliferação massiva de cianobactérias (bloom) em reservatórios de água doce superficial tem impactos no ecossistema aquático, nos procedimentos operativos das estações de tratamento de água e acarreta riscos para a saúde humana e animal devido à capacidade toxigénica de algumas espécies cianobacterianas.
Neste trabalho descreve-se a deteção de uma densidade elevada da espécie Dolichospermum planctonicum numa amostra de água tratada, bem como a presença de anatoxina-a (neurotoxina).
A legislação portuguesa apenas contempla um valor paramétrico de referência para microcistinas (hepatotoxinas). Porém, de acordo com os valores-guia da Organização Mundial da Saúde, a anatoxina-a foi detetada numa concentração abaixo do valor recomendado para água de consumo humano.
Os resultados alertam para a importância da monitorização regular de cianobactérias em água bruta e tratada e da adequação da monitorização de cianotoxinas em função do potencial tóxico das espécies presentes. Por outro lado, salienta-se a necessidade de integrar os dados de monitorização a nível do país, pois só assim será possível conhecer o cenário real de ocorrência de blooms tóxicos em Portugal, e, em conformidade, adequar a legislação nacional.Monitoring the quality of water for human consumption regarding the presence of cyanobacteria is one of the attributions of the Department of Environmental Health of the National Institute of Health (Portugal ). The massive proliferation of cyanobacteria (bloom) in freshwater bodies has impacts on aquatic ecosystems, on operative procedures at water treatment stations and presents human and animal health risks due the toxigenic potential of some cyanobacterial species.
In this work, we describe the detection of a high density of the cyanobacterium Dolichospermum planctonicum in a treated water sample, as well as the presence of anatoxin-a (neurotoxin).
Portuguese legislation only includes a mandatory level for microcystins (hepatotoxins). However, according to the World Health Organization guidelines, anatoxin-a was detected at a concentration below the recommended value for drinking water.
These results alert to the importance of the regular monitoring of cyanobacteria in raw and treated water and to the adequacy of cyanotoxins monitoring according to the toxic potential of the species present in the samples.
Furthermore, the need to integrate data at the country level is emphasized, as this is the only way to understand the real scenario of toxic bloom occurrences in Portugal and, accordingly, adapt national legislation
Distribution of Chlamydia trachomatis ompA-genotypes over three decades in Portugal
No full textObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990–2021) in Portugal.
Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed.
Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens.
Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.Objetivos: Chlamydia trachomatis é classificada em 15 genótipos principais, de A a L3, com base na diversidade do gene ompA. Neste estudo, avaliamos e caracterizamos a distribuição e diversidade dos genótipos ompA ao longo de 32 anos (1990-2021) em Portugal.
Métodos: A coleção do Laboratório Nacional de Referência para Infeções Sexualmente Transmissíveis de Portugal inclui 5824 amostras positivas para C. trachomatis que foram genotipadas com sucesso para ompA entre 1990 e 2021. Foi realizada uma análise aprofundada da distribuição dos genótipos ompA ao longo dos anos, assim como por sexo biológico, idade e local anatómico de infeção.
Resultados: O genótipo-ompA E foi consistentemente o mais frequentemente detetado ao longo dos anos, com uma frequência mediana de 34.6%, seguido pelos genótipos D/Da (17.6%), F (14.3%) e G (10.7%). A prevalência dos genótipos LGV
(L2, 62.0%, seguido por L2b, 32.1%) aumentou a partir de 2016, atingindo o pico em 2019 (20.9%). Os genótipos LGV, G e Da foram associados ao sexo biológico, nomeadamente com ser homem, e foram os mais frequentes entre as amostras anorretais (37.7%, 19.4% e 17.7%, respetivamente). Os genótipos-ompA LGV representaram 38.9% das amostras anorretais masculinas desde 2016, e também foram detetados em amostras orofaríngeas e urogenitais. O genótipo-ompA E foi o mais frequentemente detetado na orofaringe (28.6%) e na região urogenital (33.9%) durante o período do estudo, seguido por D/Da (17.4%) e F (16.0%) nas amostras urogenitais, e por G (26.1%) e D/Da (25.7%) nas amostras orofaríngeas. Os nossos dados também destacam o surgimento da estirpe recombinante L2b/D-Da a partir de 2017 (representando entre 2.0% a 15.5% dos casos de LGV por ano) e a deteção não negligenciável do genótipoompA B em amostras urogenitais e anorretais.
Conclusões: Este estudo fornece um panorama abrangente da vigilância molecular de C. trachomatis em Portugal, enfatizando não só a relevância contínua da genotipagem-ompA como complemento aos testes rápidos de deteção específicos para LGV, mas também contribuindo para um conhecimento mais profundo sobre a epidemiologia, diversidade e patogenicidade de C. trachomatis.The work by MA is partially financed by national funds through Fundação para a Ciência e a Tecnologia under the project UIDB/00006/2020 (https://doi.org/10.54499/UIDB/00006/2020
Estudos Funcionais de Variantes no Gene APOB em Doentes com Diagnóstico Clínico de Hipercolesterolemia Familiar
No full textDissertação de mestrado em Bioquímica e Biomedicina, apresentada à Faculdade de Ciências da Universidade de Lisboa, 2025Orientadora Mafalda Bourbon e coorientadora Ana Catarina Alves (Departamento de Promoção da Saúde e Prevenção de Doenças Não Transmissíveis, INSA)A hipercolesterolemia familiar (FH) é uma doença caracterizada por níveis elevados de colesterol LDL, que se acumulam nas artérias, promovendo o desenvolvimento precoce de eventos cardiovasculares. Geneticamente, a FH é transmitida de forma autossómica semi-dominante, sendo causada por variantes nos genes LDLR, APOB e PCSK9. As variantes no gene APOB afetam a ligação do colesterol LDL ao seu recetor, representando 5 a 10% dos casos de FH. No entanto, para a maioria das variantes identificadas, o efeito funcional na proteína ainda não está determinado. Esta dissertação teve como objetivo principal investigar a correlação entre fenótipo e genótipo, além de caracterizar funcionalmente variantes identificadas no gene APOB em indivíduos com diagnóstico clínico de FH na população portuguesa. Como objetivo secundário, foi desenvolvida uma base de dados que reúne todas as variantes deste gene com estudos funcionais realizados até ao momento. A identificação das variantes foi realizada por sequenciação de nova geração (NGS) e confirmada por PCR e sequenciação de Sanger, seguida da pesquisa da variante nos familiares dos casos index. As variantes em estudo foram identificadas no âmbito do projeto EPHF. O LDL dos participantes foi isolado por ultracentrifugação a partir do soro de casos index e familiares com variantes no gene APOB, bem como de indivíduos normolipidémicos. Este LDL foi marcado com fluorescência e incubado em células CHO-ldlΔ7 para avaliar a capacidade de ligação e internalização do LDL. A caracterização funcional, realizada por citometria de fluxo com LDL fluorescente, revelou que as variantes p.(Asp1456Asn), p.(Val4295Leu) e p.(Arg4519Thr) apresentavam ligação e internalização normais do LDL, indicando que estas variantes não comprometem a função da proteína. Este trabalho destaca a importância de caracterizar funcionalmente as variantes no gene APOB para melhorar o diagnóstico dos indivíduos com FH, possibilitando tratamentos mais adequados a cada caso e contribuindo para a redução do risco cardiovascular.Familial hypercholesterolemia (FH) is a disease characterized by elevated LDL cholesterol levels, which accumulate in the arteries, leading to the early onset of cardiovascular events. Genetically, FH is caused by variants in the LDLR, APOB, and PCSK9 genes, presenting a semi-dominant autosomal transmission pattern. Variants in the APOB gene affects the binding of LDL cholesterol to its receptor, accounting for 5 to 10% of FH cases. However, for most identified variants, the functional effect on the protein remains undetermined. The primary aim of this dissertation was to investigate the correlation between phenotype and genotype and to functionally characterize variants identified in the APOB gene in individuals with a clinical diagnosis of FH in the Portuguese population. A secondary objective was to develop a database that compiles all known variants of this gene with functional studies conducted to date. The identification of variants was performed using next-generation sequencing (NGS) and confirmed by PCR and Sanger sequencing, followed by screening the variants in the relatives of the index cases. The variants studied were identified as part of the EPHF project. The LDL of the participants was isolated by ultracentrifugation from the serum of index cases and relatives with variants in the APOB gene, as well as from normolipidemic individuals. This LDL was fluorescently labeled and incubated in CHO-ldlΔ7 cells to assess LDL binding and internalization capacity. Functional characterization, performed using flow cytometry with fluorescently labeled LDL, revealed that the variants p.(Asp1456Asn), p.(Val4295Leu), and p.(Arg4519Thr) exhibited normal LDL binding and internalization, indicating that these variants do not impair protein function. This study highlights the importance of functionally characterizing variants in the APOB gene to improve the diagnosis of individuals with FH, enabling more appropriate treatments for each case and contributing to the reduction of cardiovascular risk
Signature cytokine-associated transcriptome analysis of effector γδ T cells identifies subset-specific regulators of peripheral activation
No full textγδ T cells producing either interleukin-17A (γδ cells) or interferon-γ (γδ cells) are generated in the mouse thymus, but the molecular regulators of their peripheral functions are not fully characterized. Here we established an Il17a-GFP:Ifng-YFP double-reporter mouse strain to analyze at unprecedented depth the transcriptomes of pure γδ cell versus γδ cell populations from peripheral lymph nodes. Within a very high fraction of differentially expressed genes, we identify a panel of 20 new signature genes in steady-state γδ cells versus γδ cells, which we further validate in models of experimental autoimmune encephalomyelitis and cerebral malaria, respectively. Among the signature genes, we show that the co-receptor CD6 and the signaling protein Themis promote the activation and proliferation of peripheral γδ cells in response to T cell antigen receptor stimulation in vitro and to Plasmodium infection in vivo. This resource can help to understand the distinct activities of effector γδ T cell subsets in pathophysiology.The project leading to these results has received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement number 646701 to B.S.-S.), ‘la Caixa’ Foundation under project code LCF/PR/HR24/00929 (to B.S.-S.), Fundação para a Ciência e Tecnologia, Ministério da Ciência, Tecnologia e Ensino Superior, Portugal (PTDC/MED-IMU/32296/2017 to A.M.C.; SFRH/BD/145352/2019 to D.I.; Decree-law number 57/2016, as amended by the Law number 57/2017 to A.P.) and Fondation pour la Recherche Médicale (to R.L.)
Unveiling the Role of APOB Variants in Familial Hypercholesterolemia: Functional Insights
No full textFamilial hypercholesterolemia (FH) is a condition characterized by increased LDL cholesterol levels with APOB variants accounting for about 5-10% of FH cases. However, variants in this gene may be more common than initially estimated since the entire APOB gene has only recently started to be sequenced. Although most of the alterations are missense, nonsense variants and small indels in exon 29 were also identified in individuals with FH phenotype and can be the cause of disease. This work aimed to characterize APOB variants identified in individuals with clinical diagnosis of FH. Moreover, we intended to do an overview of the APOB variants presenting functional studies.
PubMed repository was consulted to collect publications regarding functional characterization of APOB variants. For variant characterization, LDL was isolated through sequential ultracentrifugation. ED-LDLR fragments purified from HEK293 cells were incubated with the different APOB variants and antibodies, to determine apoB affinity for LDLR by ELISA assay. CHO-ldlA7 cells were transfected with wt LDLR plasmid and incubated with FITC-labeled LDL to determine LDL binding and uptake by flow cytometry.
In the literature there are 23 APOB variants with functional studies, six of which characterized by our group. Fourteen variants affecting apoB normal function; the remaining results presenting normal apoB function. Recently we characterized 8 more variants: p.(Ala1393Val), p.(Asp1456Asn), p.(Met2042Thr), p.(Asp2213del), p.(Ile3374Thr), p.(Val4295Leu) and p.(Arg4519Thr) that do not appear to impact apoB's binding to the LDLR; p.(Gln4316*) demonstrated reduced affinity for the LDL receptor.
Functional studies play a critical role in assessing the pathogenicity of genetic variants and are among the key criteria for variant classification. These in-depth analyses confirm clinical diagnosis and provide essential insights for developing personalized treatment strategies. In the future, we aim to increase the number of studied variants, starting with 15 more variants from the Portuguese FH Study.Personalizing diagnosis and treatment for Familial Hypercholesterolaemia patients (PerMedFH) project (HR23-00749 (BPD2)) funded by La Caixa Foundation in partnership with FCT
Rare serotype c Haemophilus influenzae invasive isolate: characterization of the first case in Portugal
No full textBrief ReportWe report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype.info:eu-repo/semantics/publishedVersio
Regulatory Variants In LDLR And PCSK9 Promoters And 5'UTRs: Investigating The impact In Familial Hypercholesterolaemia
No full textSessão da apresentação - SaaG Session: Genetic insights in FH and Dyslipidemia.Background and Aims: Familial Hypercholesterolaemia (FH) is a genetic disorder of lipid metabolism caused by pathogenic variants in LDLR, APOB, and PCSK9. While
diagnostic efforts traditionally focus on coding variants, non-coding regions, such as promoters and 5'UTRs, remain understudied despite their importance. This work aims
to characterise 100 variants in the promotor/5'UTR of LDLR and PCSK9. Methods: The promotor/5'UTR sequences of LDLR and PCSK9 were cloned by SOEing PCR upstream of the Firefly luciferase coding region in the pGL4.10. For LDLR, sequence from c.-319 to the initiation codon was retrieved from literature, while for PCSK9, sequence from c.-650 to the initiation codon was confirmed using a 5'-RACE strategy. The resulting constructs (LDLR_pGL4-WT and PCSK9_pGL4-WT) were subsequently modified through site-directed mutagenesis. LDLR and PCSK9 variants were transfected into CHO-ldlA7 and Huh7 cells,
respectively. Cells were cultured in different cholesterol depletion states, and luciferase activity measured using a Dual-Luciferase Reporter Assay System.
Results: Compared to their respective wild-type constructs, LDLR and PCSK9 variants displayed a diverse range of phenotypic effects, with statistically significant
increases or decreases in promoter activity. These variations can differently impact the FH phenotype and hold significant implications for disease management and
therapeutic strategies, as increases or decreases in promoter activity in the two genes have distinctly opposing effects on LDL-C levels. Moreover, as far as we know, this is the
first experimental work defining the PCSK9 5’ UTR region.
Conclusions: This study provides novel insights into the functional impact of LDLR and PCSK9 promoter/5'UTR variants on gene expression and their potential
contributions to the FH phenotype. Importantly, these findings underscore the critical role of functional studies in variant classification, particularly for non-coding regions,
which remain underrepresented in genetic diagnostics. By elucidating how these variants influence LDL-C levels through altered promoter activity, this work highlights
their relevance in refining FH diagnosis and tailoring patient management strategies.Personalizing diagnosis and treatment for Familial Hypercholesterolaemia patients (PerMedFH) project (HR23-00749 (BPD2)) funded by La Caixa Foundation in partnership with FCT
Effectiveness of the XBB.1.5 COVID-19 Vaccines Against SARS-CoV-2 Hospitalisation Among Adults Aged ≥ 65 Years During the BA.2.86/JN.1 Predominant Period, VEBIS Hospital Study, Europe, November 2023 to May 2024
No full textVEBIS SARI VE network team - Portugal: Ana Paula Rodrigues, Débora Pereira, Margarida Tavares, Susana Costa Maia e Silva, Paula Pinto, Cristina Bárbara, António Pais de Lacerda, Raquel Guiomar, Camila Henriques.We estimated the effectiveness of the adapted monovalent XBB.1.5 COVID-19 vaccines against PCR-confirmed SARS-CoV-2 hospitalisation during the BA.2.86/JN.1 lineage-predominant period using a multicentre test-negative case-control study in Europe. We included older adults (≥ 65 years) hospitalised with severe acute respiratory infection from November 2023 to May 2024. Vaccine effectiveness was 46% at 14-59 days and 34% at 60-119 days, with no effect thereafter. The XBB.1.5 COVID-19 vaccines conferred protection against BA.2.86 lineage hospitalisation in the first 4 months post-vaccination.The ‘Vaccine Effectiveness, Burden and Impact Studies’ (VEBIS) is a project of the European Centre for Disease Prevention and Control (ECDC) run under the framework contract No. ECDC/2021/016 (EU-H)
Physiologically based toxicokinetic models in aggregate exposure: A review
No full textThis literature review explores the application of Physiologically Based Kinetic (PBK) models in aggregate exposure (AE) assessment across different chemical classes. It builds on the screening of 1119 publications and the identification of 40 relevant articles. The most frequently studied chemicals include volatile organic compounds and plant protection products, with metals, personal care products, persistent organic pollutants and plasticisers also represented. Most studies reported in this review are applied to human populations and build on human biomonitoring (HBM) data to enhance model reliability. However, some studies use animal models (primarily rat models) and apply cross-species extrapolation to the human AE scenario. Occupational exposure is taken into consideration as part of the AE scenario in a few studies. Many of the reviewed studies are designed in support of chemical risk assessment (CRA), illustrating the wide applicability of PBK models. The review discusses the joint role of HBM data and PBK model in AE scenarios, highlighting its importance for a reliable risk assessments. The studies identified and discussed in this review suggest a broad interpretation of AE. The diversity across case reported studies is attributed to varying interpretations and existing definitions of AE. Finally, the roles of forward and reverse dosimetry in refining AE assessments are discussed, highlighting their importance for future research. This scoping review provides a comprehensive overview of PBK model applications in addressing AE, serving as a valuable foundation for future research and development aimed at advancing human health protection towards the Next-Generation Risk Assessment (NGRA).Highlights: - Aggregate exposure is key to comprehensive chemical risk assessment: - Physiologically based kinetic models are applied to aggregate exposure using diverse approaches; - The use of human biomonitoring data and physiologically based kinetic models can enhance aggregate exposure and risk assessment.This research work was partially funded by the European Food Safety Authority (EFSA) under the contract agreement (OC/EFSA/ED/2022/04) to deliver the ExpoAdvance roadmap